C-reactive protein: An inflammatory marker with specific role in physiology, pathology, and diagnosis

Introduction C-reactive protein (CRP) is one of the common test parameters used in clinical practice, to assess, diagnose, and prognose inflammation. However, the role played by CRP in physiological processes is not clearly elucidated. CRP, belonging to pentraxin family of proteins shows a 1000-fold or more increase in concentration during the occurrence of an injury, inflammation or tissue death.1 The plasma half-life of CRP is about 19 hours and is constant under all conditions of health and disease.2, 3 In addition to CRP, the levels of few other proteins termed as acute phase proteins (APR) are also increased during inflammation. CRP, the first acute-phase protein to be described, is a sensitive systemic marker of inflammation and tissue damage.2, 4 Precise response and ease of assay have made CRP an ideal marker of inflammation. CRP was discovered in 1930 by William Tillett and Thomas Francis of Rockefeller University.5 The researchers have reported that a third serologic fraction or ‘fraction C’ isolated from pneumococcus infected patients, was different from capsular polysaccharide and nucleoprotein fractions detectable by specific antibody response.5 Oswald Avery and Maclyn McCarty, who postulated the ‘transforming principle’ and the concept that genes are made of DNA, also described that CRP as an ‘acute-phase reactant’ that was found to be increased in serum of patients having a spectrum of inflammatory stimuli.6, 7 Volanakis and Kaplan later identified the specific ligands that bind to CRP.8 CRP was found to have the function of conjugating pathogens and inducing their destruction by complement system. It was also studied as a screening marker of inflammation, disease activity, and as a diagnostic adjunct.9 The exact role of CRP as an acute phase protein needs to be evaluated further. This review focuses on the diagnostic significance of CRP in various disease conditions as well as the established and controversial evidence on its physiological role.


Introduction
C-reactive protein (CRP) is one of the common test parameters used in clinical practice, to assess, diagnose, and prognose inflammation. However, the role played by CRP in physiological processes is not clearly elucidated. CRP, belonging to pentraxin family of proteins shows a 1000-fold or more increase in concentration during the occurrence of an injury, inflammation or tissue death. 1 The plasma half-life of CRP is about 19 hours and is constant under all conditions of health and disease. 2,3 In addition to CRP, the levels of few other proteins termed as acute phase proteins (APR) are also increased during inflammation. CRP, the first acute-phase protein to be described, is a sensitive systemic marker of inflammation and tissue damage. 2,4 Precise response and ease of assay have made CRP an ideal marker of inflammation. CRP was discovered in 1930 by William Tillett and Thomas Francis of Rockefeller University. 5 The researchers have reported that a third serologic fraction or 'fraction C' isolated from pneumococcus infected patients, was different from capsular polysaccharide and nucleoprotein fractions detectable by specific antibody response. 5 Oswald Avery and Maclyn McCarty, who postulated the 'transforming principle' and the concept that genes are made of DNA, also described that CRP as an 'acute-phase reactant' that was found to be increased in serum of patients having a spectrum of inflammatory stimuli. 6,7 Volanakis and Kaplan later identified the specific ligands that bind to CRP. 8 CRP was found to have the function of conjugating pathogens and inducing their destruction by complement system. It was also studied as a screening marker of inflammation, disease activity, and as a diagnostic adjunct. 9 The exact role of CRP as an acute phase protein needs to be evaluated further. This review focuses on the diagnostic significance of CRP in various disease conditions as well as the established and controversial evidence on its physiological role.

Production of CRP
CRP is produced in many sites within the human body ( Figure 1). It is produced in the liver in response to IL-6. Products of activated monocytes in Hep 3B cells induce the production of human serum amyloid A (SAA) protein and CRP, but not by IL-1β, TNF-α, or some hepatocytestimulating factor preparations. It is also produced in very limited concentration by non-hepatic cells like neurons, atherosclerotic plaques, monocytes, Kupffer cells and lymphocytes. 1,10,11 Studies have shown that epithelial cells of both respiratory tract and renal epithelium can also produce CRP under certain circumstances. 12,13 Recent studies have demonstrated that human coronary artery smooth muscle cells could also synthesize CRP upon stimulation by inflammatory cytokines. 14,15 Cogent data have indicated that the protein is also produced by the atherosclerotic lesions (especially by smooth muscle cells and macrophages), kidneys, neurons, and alveolar macrophages. 16 Additionally, there is evidence to suggest that lipid peroxidation and infection, such as cytomegalovirus may trigger a pro-inflammatory cytokine cascade resulting in CRP release. 17 CRP may be secreted from active human peripheral blood monocytes while generation from peripheral blood mononuclear cells (PBMC) is poorly established. 12,14,18 Expression of CRP by human respiratory epithelial cells and alveolar macrophages suggests contribution to bacterial clearance and direct involvement in pulmonary host defense and immune response. 11,19 Biosynthetic labeling with S-met and immuno-precipitation with anti-CRP antibodies and Staphylococcus aureus indicate that cell surface CRP is produced by lymphocytes. 11,20,21,35 Structure of CRP CRP is a pattern recognition molecule binding to specific molecular configurations that are typically exposed during cell death or found on the surfaces of pathogens. It is a calcium-dependent ligand-binding plasma protein, which is phylogenetically highly conserved with homologues in vertebrates and many invertebrates. 1 Human CRP is a non-glycosylated polypeptide with five identical subunits or protomers. 5 Each subunit is constituted by 206 amino acid residues and bound to each other by non-covalent bonds. 2 Structure of CRP based on the amino acid composition, as derived from the sequence data and a minimal molecular weight of 20,946, has been calculated for human CRP. 1 X-ray crystallography has demonstrated the structure of the protomer ( Figure 2) as two antiparallel β-sheets with a flattened jelly-roll topology similar to that of lectins, especially concanavalin. 1,6,7 Each subunit has a recognition face with a phosphocholine binding site consisting of two coordinated calcium ions adjacent to a hydrophobic pocket. Phe-66 and Glu-81 are the two key residues mediating the binding of phosphocholine to CRP. Phe-66 provides hydrophobic interactions with the methyl groups and Glu-81 is found on the opposite end of the pocket where it interacts with the positively charged nitrogen of phosphocholine. 1 The opposite face of the pentamer is the effector face, where complement C1q binds and serves as Fcγ receptors. CRP binding to C1q activates the classical complement pathway up to the level of the C3 convertase. The role of CRP receptors in various processes is mapped in figure 3.

Functions of CRP
CRP, an inducible protein secreted in response to inflammatory stimulus, binds to pathogens and activates the complement to enhance opsonisation and clearance, even before the production of specific IgM or IgG. Involvement of CRP in various immunological processes is mapped in figure 4. CRP bound to a multivalent ligand initiates the assembly of a C3 convertase through classical pathway, which leads to the presentation of ligand with opsonic Binds to bacteria or cells and interacts with the natural killer cells and monocytes, may increase the tumoricidal activity of these cells 5,11 Activates endothelial cells to express adhesion molecules, chemokines, and cytokines Inhibits nitric oxide (NO) production and stimulation of nitric oxide release through down regulation of endothelial nitric oxide synthase Upregulates angiotensin receptor-1 (AT1-R) protein expression, increases AT1-R number on vascular smooth muscle cells, and promotes vascular smooth muscle migration and proliferation in vitro Serves as chemoattractant for monocytes and induces tissue factor expression in macrophages Mediates uptake of native LDL into macrophages 14,27 Activates complement pathway up to C5 convertase and enhances phagocytosis Amplifies inflammatory responses 5,11 22 However, the protein does not favor the formation of a C5 convertase and hence, CRPinitiated complement activation does not mediate acute inflammatory reactions and membrane damage . 22 The protein has been shown to induce the synthesis of IL-1α, IL-1β, TNF-α, and IL-6 in human peripheral blood mononuclear cells and alveolar macrophages. 5 Furthermore, soluble and immobilized CRPs have been demonstrated to mediate the uptake of native low density lipoprotein (LDL) into macrophages. 23 CRP may also function as a substrate for membrane-associated neutrophil serine protease that cannot be up-regulated. On the contrary, the degradation of CRP yields small soluble bioactive peptides ( Figure  5) that inhibit many of the pro-inflammatory and tissuedestructive potential of neutrophils. These peptides are possibly involved in signal transduction pathways leading to neutrophil activation. 4 CRP shares major amino acid sequences with SAA fragments. Heat-aggregated CRP has been demonstrated to activate platelet aggregation, secretion, and generation of thromboxane A2, similar to heat-aggregated IgG. Human SAA seems to selectively modulate platelet reactivity and down-regulate at least Three of the synthetic peptides corresponding to residues 201-206 (CRP-III), 83-90 (CRP-IV), and 77-82 (CRP-V) of the intact protein were identified to act additively to inhibit superoxide production from activated neutrophils at 50 µM, whereas CRP-III and CRP-V inhibit neutrophil chemotaxis. 24,25 Studies indicate a specific activationindependent action of CRP, CRP peptides (174-185), and CRP-III on the expression of L-selectin. CRP peptides attenuate neutrophil adhesion to the endothelium and consequently neutrophil trafficking into tissues, thereby limiting the inflammatory response. 26 Some of the critical functions of CRP are shown in table 1.

Ligand interactions
CRP is the first pattern recognition receptor identified possessing greater affinity to bind to a molecule identified by a specific pattern. 28 The ligand binding site of CRP, composed of loops with two calcium ions 4 Å apart and bound by protein side-chains, is located on the concave face. 5 Phosphocholine (PC), a component in the biological cell membranes of bacteria and fungi, is the first identified ligand that binds to CRP. 1, 2 Ligands known to bind to CRP are listed in table 2.

CRP gene regulation
The CRP gene, located on the1q23.2 on chromosome 1, contains one intron separating the region encoding the signal peptide from that encoding the mature protein. 1,29 The CRP gene sequence was determined in 1985 simultaneously by two different research teams. 30,31 The first exon encodes a signal peptide and the first two amino acids of the mature protein. This is followed by a 278-nucleotide-long intron that includes a GT repeat sequence. The second exon encodes the remaining 204 amino acids, followed by a stop codon. 32 Goldman et al. has reported for the first time that the GT stretch in the intron is polymorphic in length. Two recent studies describe polymorphisms in the CRP intron gene and promoter that influences the normal expression levels. 33 Individuals with particular allele combinations exhibit two-fold lower baseline CRP levels, perhaps due to DNA structural changes that affect transcription. 33 Within the promoter, several polymorphisms were discovered in transcription factor binding E-box sites, all of which Autologous ligands include native and modified plasma lipoproteins, damaged cell membranes, different phospholipids and related compounds, small nuclear ribonucleoprotein particles, and apoptotic cells. 34,35,36 Extrinsic ligands include many glycan, phospholipid, and other constituents of microorganisms, such as capsular and somatic components of bacteria, fungi, and parasites, as well as plant products.
Macromolecular ligands, phosphoethanolamine, chromatin, histones, fibronectin, small nuclear ribonucleoproteins, laminin, and polycations 20,37 Phosphocholine esters, polycations and galactans (lectin character) 34 In vitro, human CRP directly inhibits the binding of leptin to its receptors and blocks its ability to signal in cultured cells. Human CRP has been correlated with increased adiposity and plasma leptin. Thus, suggesting a potential mechanism contributing to leptin resistance by which circulating CRP binds to leptin and attenuates its physiological functions. 38 Phosphorylcholine (Ca2+-dependent binding specificity), lipids (polar head group), lecithin (phosphatidylcholine, PC) and sphingomyelin (SM), and plasma lipoproteins 34 Histones H1 and H2A most strongly and less binding to H2B, H3 and H4, and polycations 37

CRP
Neutrophil oxidative capacity (Interaction with receptors and direct anti-ox activity)

Down regulates
Complement factor c1q7, regulatory protein factor H and C4B binding protein (C4BP)

Binds to
Protein synthesis of IL-1α, IL-1β and TNF-α

Formation of monocyte platelet aggregates (ex vivo and in vivo)
Classical complement pathway

Provides
Induces Affect resulted in different baseline circulating levels of CRP and response by other genes that encode cytokines affecting its synthesis, such as IL-6, IL-1 and TNF-α. 29 Single nucleotide polymorphisms (SNPs) across the CRP gene have been associated with differences in basal CRP levels. CRP gene contains binding sites for STAT3 (transcription factors) and Rel proteins. 1 It is well established that IL-6 stimulates the acute phase expression of CRP. 39 Polymorphism in the human CRP gene resulting in a lower basal level of CRP has been associated with an increased risk of developing systemic lupus erythematosus. 1

Different functional forms of CRP
Studies have found different structural forms of CRP ( Figure 6), such as, the pentameric ring, globulin and fibril structures, which are observed by combination of size-exclusion chromatography and electron microscopy. Denatured and aggregated forms of CRP (neo-CRP or modified CRP) have been also reported. 40 The pentameric ring-like CRP was observed mostly on ligand containing membrane in a calcium-dependent manner. The globulin-like monomers, found on negatively charged membrane in the absence of calcium, exhibit structural stability. The fibril-like structures were formed by face-to-face stacking of a number (several to hundreds) of pentameric CRP. The freshly purified CRP forms short single-strand fibrils while that stored for more than several days form long and bundled fibrils. In 1965, Gotschlich and Edelman reported for the first time that the CRPs purified from serum were mainly pentamers. 41 CRP exists in two distinct forms: (i) native pentameric CRP (nCRP), detectable in serum with both pro-and anti-inflammatory effects, and (ii) the tissue-bound modified or monomeric CRP (mCRP), with predominantly pro-inflammatory effects. 42 Native CRP, which exists as a pentamer, dissociates to mCRP due to conformational rearrangement. There is growing evidence that mCRP may have novel proinflammatory and thrombotic properties. 43 mCRP is found to be deposited in human aortic and carotid atherosclerotic plaques, but not in healthy vessels. 44 In 1983, Potempa et al. reported another type of CRP, termed 'modified CRP',

Figure 5: Degradation products of CRP and their functions
produced on urea-EDTA or acid-EDTA treatment. 45 The modified CRP runs faster in gel electrophoresis and has lower solubility than native CRP. 40 pCRP is formed by urea-chelation treatment and resembles the free subunit mCRP. mCRP has distinct physicochemical, antigenic and biologic activities compared to CRP. 46 mCRP enhances platelet aggregation and secretion of serotonin, modulation of arachidonic acid metabolism, stimulation of interleukin-1 (IL 1) release, potentiation of the respiratory burst response of human neutrophils, and peripheral blood monocytes to heat modified IgG. 4 The different structural forms may convert to each other under certain conditions, suggesting structural basis of multiple functions of CRP. Native CRP binds to CD32, whereas mCRP binds to CD16. It was suggested that native CRP dissociated into monomeric units on binding to plasma membrane, or in a denaturing or oxidative environment. 14,40 mCRP can inhibit as well as activate the classical complement pathway by binding to C1q, depending on its presence in a fluid phase or surface-bound state. 48 Identification of suitable assays that allow direct testing of mCRP, instead of native CRP in serum or tissue, will further clarify its biological significance. 14 Role of CRP in physiology and pathology CRP, mainly recognized as a biomarker of inflammation, is now viewed as a direct contributor in atherosclerosis as it functions both as 'pro-inflammatory' and 'anti-inflammatory' molecule. 1 With the advent of high-sensitivity assays for determining CRP, the protein has emerged as one of the most powerful independent predictors of cardiovascular disease. CRP level, which significantly increases in acute coronary syndromes, has a prognostic value in patients with cardiovascular complications and in apparently healthy individuals. The in vivo mechanisms of CRP as a mediator of the inflammatory state and thrombotic complications are continuing to be unraveled. Here, we focus on the role of CRP in the pathophysiology of atherosclerosis including the potential mechanisms of action in circulation as well as the potential contribution of genetic variations within the CRP gene. 49 The capacity of human CRP to activate/ regulate complement may be an important characteristic that links CRP and inflammation with atherosclerosis. Recent advances suggest that, in addition to classical pentameric CRP, mCRP may also play an active role in atherosclerosis. The capacity of mCRP to interact and activate the complement cascade is unknown. 48 Loss Storage form of pentameric symmetry in CRP is associated with the appearance of novel bioactivities in mCRP that enhance neutrophil localization and activation at the inflamed or injured vascular sites. 50 The biological effect of CRP on the development of atherosclerosis seems to encompass a complex network of interactions with other players in immunity and inflammation, such as the complement system. It may also involve the direct effect of CRP on the cells involved in lesion growth and development. 51 Evidence suggests that CRP functions as a powerful proatherogenic factor, in addition to being a risk factor for atherosclerotic and metabolic events. A growing body of evidence implicates CRP as a powerful risk marker for diverse cardiovascular and metabolic diseases. Possibly, it is also a mediator of these diseases as it contributes to the substrate underlying lesion formation, plaque rupture, and coronary thrombosis. 17,51 A CRP mutant incapable of binding to PC provides a tool to assess PC-dependent interactions of CRP with the other biologically significant ligands and to further investigate the functions of CRP in host defense and inflammation. 52 Findings indicate that CRP can modify the course of autoimmune disease, possibly by preventing the exposure of nuclear antigens to the immune system. A close relationship between leptin and CRP supports the view that the adipokine (leptin) has a possible role in inflammation and atherothrombosis, besides being involved in the pathophysiology of obesity. 38,53 Physiological concentrations of leptin can stimulate expression of CRP in human primary hepatocytes. Recently, human CRP has been correlated with increased adiposity and plasma leptin, suggesting that circulating CRP binds to leptin and attenuates its physiological functions. This could be a potential mechanism contributing to leptin resistance.
A growing body of evidence implicates CRP as a direct mediator of endothelial dysfunction. 54 Patients with elevated levels of CRP have been shown to elicit impaired endothelium-dependent vasodilatation, suggesting that CRP may be a useful clinical tool for endothelial vasomotion. 55 CRP may also directly promote monocyte activation by stimulating the release of cytokines, such as IL-1b, IL-6, and TNF-α. 50 Recent evidence shows that CRP is deposited in the arterial intima, at the sites of atherogenesis. 23, 56 CRP-induces up-regulation of adhesion molecules and monocyte chemoattractant protein-1 in venous endothelial cells. CRP is proatherogenic in monocyte/macrophages, because it increases tissue factor expression, promotes monocyte chemotaxis and adhesion to endothelial cells, release of reactive oxygen species and matrix metalloproteinase-1, and the uptake of oxidized low-density lipoprotein, leading to increased foam cell formation. Furthermore, CRP is present in foam cells in the atherosclerotic lesion and activates complement. 57, 58

CRP as a marker of various diseases/conditions
In the absence of inflammation, CRP is not constitutively expressed and its level is undetectable. Baseline concentrations of CRP are influenced by many factors, including chronic microbial infections, smoking, BMI, coffee consumption, oral contraceptive use, and genetics. 57 Lifestyle factors, such as smoking and BMI, have a greater influence on baseline CRP levels than single nucleotide polymorphisms (SNPs), making the identification of a genetic association of CRP SNPs with cardiovascular diseases difficult. 59 The level of CRP is altered in variety of conditions; although the rise in CRP is non-specific, the quantum and the pattern of rise will help deduce the diagnosis. A few clinical situations are discussed in the following sections.
CRP during normal pregnancy CRP does not cross the placental barrier and therefore, will be useful in diagnosing infections in newborns. 60 Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and the elevated levels are associated with adverse pregnancy outcome. 61 These results demonstrate that the human placenta produces and releases CRP, like other placental proteins, mainly into the maternal circulation.

CRP and cardiovascular risk
The association between CRP and cardiovascular risk is driven predominantly by systemic inflammation (Table  3). CRP is unlikely to contribute directly to cardiovascular disease as a pathogenic factor. Similar conclusions were drawn from recent Mendelian randomization studies. Using widely available high-sensitivity assays, CRP levels of 1, 1 to 3, and 3 mg/L have been classified as low, moderate, and high-risk groups for future cardiovascular events. Individuals with LDL cholesterol below 130 mg/dL and CRP levels of 3 mg/dL represent a highrisk group. The conversion of plasma CRP (pCRP) to monomeric CRP (mCRP) has been described as being mediated by activated platelets, which are associated with cardiovascular risks. 3,44 Sl. No.

Advantages Disadvantages References
Coronary heart disease (CHD) The Women's Health Study (WHS) did not find an association between lipoproteinassociated phospholipase A 2 (Lp-PLA 2 ) mass and cardiovascular disease, although CRP was significantly associated with disease incidence. CRP is associated with stroke severity and long-term mortality when measured at least 24 hours after onset. Crude association between high CRP and short term functional outcome, which is likely secondary to stroke severity. CRP is an independent predictor of long-term mortality after ischemic stroke.     suggests bacterial infections, whereas that below 10 mg/L indicates viral infection. In tuberculosis, it is often found to be between 10 to 100 mg/L. 66 Additional determination of procalcitonin can add specificity in the case of bacterial infections. 67 The above information is also helpful to distinguish infection from an autoimmune flare. Similarly, the rate of change in CRP levels can differentiate tuberculosis from bacterial pneumonia. 68

CRP and inflammatory diseases
In the case of inflammatory diseases, CRP level represents the disease activity. Studies have suggested direct correlations of CRP with RA and inflammatory bowel diseases like Crohn's disease. 69,70 In contrast, in conditions like SLE, CRP is not significantly elevated.

CRP and obesity
CRP concentrations are elevated, predominantly in obese individuals who are insulin resistant, and are in line with the weight loss-associated improvements in insulin resistance. The relation between CRP concentrations and insulin resistance is independent of obesity. 47

CRP and diabetes
Elevated levels of CRP and IL-6 predict the development of type 2 diabetes. This association supports a possible High CRP, low albumins provide prognostic information independent of each other in CKD 3-4.
Both are independent risk factors for all-cause mortality.
High CRP, but not serum albumin, is a risk factor for cardiovascular mortality.

Menon-2005 143
Chronic rheumatic valve disease Low Hs-CRP was associated with a significantly more rapid cognitive decline and functional decline relative to higher levels.
Published studies tend to show the opposite; that is, high hsCRP levels were associated with cognitive decline The effects of hsCRP can be confounded by medications (NSAIDs and statins) Locascio-2008 145 Minor increase in CRP level has also been reported to be associated with a number of medical conditions that do not appear to be associated with inflammation. Elevated CRP is also observed with several genetic polymorphisms of the CRP and other genes, ethnicity, dietary patterns and obesity. 1

Conclusion
CRP is a valuable inflammatory biomarker in various clinical conditions. However, being non-specific, its use is limited. Concomitant occurrence of multiple stimuli of inflammation, and influence of factors other than inflammation, like smoking, obesity and physical stress, reduce the specificity of CRP significantly. In view of these, guidelines are necessary to interpret the CRP levels in a clinical context. Standardization of measurement techniques and reporting should improve the utility of CRP in regular clinical practice.