Neutrophil extravasation into inflamed or injured areas involves a complex interaction of leukocytes with endothelial cells *via* regulated expression of surface adhesion molecules. The initial attachment of neutrophils to endothelium is mediated by L-selectin (CD62L) (Díaz-González et al., 1995; Walcheck et al., 1996). L-Selectin is constitutively expressed by

antiangiogenic and antitumour activity in its cleaved conformation.

**4.2 Cleaved forms of AAPs**

modified LDL (Singh & Fudenberg, 2009).

**4.2.1 CRP**

al., 1997).

The expression of a sialyl Lewis x{sLex, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc-} portion in the carbohydrate chains of human AGP molecules has been shown to be of importance in inflammation. De Graaf et al*.* (1993) found a direct relationship between the reactivity of human AGP to the fucose-specific binding lectin (*Aleuria auranita*) and staining of human AGP by anti-sLex monoclonal antibody under healthy and disease conditions.

In a recent study a recombinant form of sialyl Lewisx(sLex)-bearing (sAGP) was administered intravenously to rats after 50 min of intestinal ischemia just before 4 h of reperfusion. A non-sLex-bearing form of AGP (nsAGP) was used as control. sAGP-treated animals had a 62% reduction in remote lung injury, assessed by 125I-albumin permeability, compared with those treated with nsAGP. There was a reduction in pulmonary myeloperoxidase levels in sAGP-treated rats compared with nsAGP-treated rats. Complement-dependent intestinal injury, assessed by 125I-albumin permeability was reduced by 28% in animals treated with sAGP compared with those treated with nsAGP leading the authors to conclude that sAGP ameliorates both complement- and neutrophilmediated injury (Williams et al., 1997).

The changes in the glycosylation pattern of AGP has been found in patients with ulcerative colitis (Ryden et al., 1997), and in patients with various liver diseases (alcoholic liver disease, hepatitis B, hepatitis C cirrhosis). For example, hyperfucosylation occurred in all cases of liver disease, although the hepatitis B and C samples showed a more significant increase in comparison with the others. Additionally N-acetylgalactosamine (GalNAc) was detected in the majority of the hepatitis C samples, which was unexpected since this monosaccharide is not a usual component of the N-linked oligosaccharide chains (Anderson et al., 2002).

In the group of Type I diabetic patients with increased urinary albumin excretion, a significant increase in alpha3-fucosylation of AGP could be detected. Therefore, the increased alpha3-fucosylation of AGP can be used as a putative marker for the development of vascular complications in Type I diabetic patients (Poland et al., 2001).

AGP and its derivatives, prepared by sequential enzymatic cleavage of the carbohydrate units, were tested for their nerve-growth-promoting activities with explants of whole dorsal root ganglia from chick embryos. The results showed that the AGP derivatives with terminal galactose, N-acetylglucosamine, or mannose have marked neurite-promoting activities (Liu et al., 1988).

#### **4.1.2 Other APPs**

AAT has a molecular weight of *M*r 52 000, and is 12% carbohydrate by weight. The AAT molecule carries a high negative charge because of sialic acid residues on the three complex glycans attached to asparagine residues 46, 83, and 247. Isoelectric focusing of plasma AAT leads to the detection of eight bands, which are numbered M1 to M8 (anodal-low pH to cathodal-high pH). The bands M4 and M6 are the most abundant of the isoforms, making up 40% and 34% of the total plasma AAT,respectively, whereas M3 and M5 are present in only trace amounts (Mills et al., 2001). Fucosylated AAT was analyzed individually and in combination with the currently used marker, alpha-fetoprotein, for the ability to distinguish between a diagnosis of cirrhosis and hepatocellular carcinoma (HCC). The levels of fucosylated AAT were significantly higher in patients with HCC compared to those with cirrhosis (Wang et al., 2009). Remarkably, non-glycosylated AAT showed shorter half life and no ability to interact with IL-8 than compared to glycosylated form of AAT (Bergin et al., 2010). This suggests an importance of AAT glycosylation for its biological activities.

ACT (alpha 1-antichymotrypsin), a serine anti-protease with specificity against neutrophil cathepsin G, is homologous with AAT, plasminogen activator inhibitor and angiotensinogen. ACT is an APP with carbohydrate content 24% of molecular weight. As for other glycoproteins, micro-heterogeneity of ACT may be ascribed to differences in carbohydrate structure, and indeed different patterns of ACT micro-heterogeneity has been shown in different diseases including cancer, heart failure and rheumatoid arthritis (Saldova et al., 2008; Kazmierczak et al.,1995; Havenaar et al., 1998). A low content of terminal GlcNac glycans and sialic acid in peripheral ACT has been suggested as a marker of progression in Alzheimer's disease (Ianni et al., 2010). The changes in patterns of glycosylation of transferrin (Tf) towards highly branched glycans have been observed in iron deficiency anaemia, rheumatoid arthritis, liver cirrhosis or in physiological state such as pregnancy. Differences in glycosylation of Tf seems to alter the metabolism of iron (Yang et al., 2005; van Pelt J et al., 1996; Dupre et al., 2001). Changes in the glycosylation pattern of major serum APPs such as Hp, AGP, AAT, Tf and alpha-fetoprotein have been recently shown in patients with pancreatic cancer and chronic pancreatitis (Sarrats et al., 2010)

#### **4.2 Cleaved forms of AAPs**

The cleaved modifications of APPs may lead to a functional deficiency of the protein, but the cleaved forms of APPs may themselves express new biological activities. For example, antithrombin which functions as an inhibitor of thrombin and other enzymes, has potent antiangiogenic and antitumour activity in its cleaved conformation.

#### **4.2.1 CRP**

40 Acute Phase Proteins – Regulation and Functions of Acute Phase Proteins

The expression of a sialyl Lewis x{sLex, NeuAcα2-3Galβ1-4(Fucα1-3)GlcNAc-} portion in the carbohydrate chains of human AGP molecules has been shown to be of importance in inflammation. De Graaf et al*.* (1993) found a direct relationship between the reactivity of human AGP to the fucose-specific binding lectin (*Aleuria auranita*) and staining of human

In a recent study a recombinant form of sialyl Lewisx(sLex)-bearing (sAGP) was administered intravenously to rats after 50 min of intestinal ischemia just before 4 h of reperfusion. A non-sLex-bearing form of AGP (nsAGP) was used as control. sAGP-treated animals had a 62% reduction in remote lung injury, assessed by 125I-albumin permeability, compared with those treated with nsAGP. There was a reduction in pulmonary myeloperoxidase levels in sAGP-treated rats compared with nsAGP-treated rats. Complement-dependent intestinal injury, assessed by 125I-albumin permeability was reduced by 28% in animals treated with sAGP compared with those treated with nsAGP leading the authors to conclude that sAGP ameliorates both complement- and neutrophil-

The changes in the glycosylation pattern of AGP has been found in patients with ulcerative colitis (Ryden et al., 1997), and in patients with various liver diseases (alcoholic liver disease, hepatitis B, hepatitis C cirrhosis). For example, hyperfucosylation occurred in all cases of liver disease, although the hepatitis B and C samples showed a more significant increase in comparison with the others. Additionally N-acetylgalactosamine (GalNAc) was detected in the majority of the hepatitis C samples, which was unexpected since this monosaccharide is not a usual component of the N-linked oligosaccharide chains (Anderson et al., 2002). In the group of Type I diabetic patients with increased urinary albumin excretion, a significant increase in alpha3-fucosylation of AGP could be detected. Therefore, the increased alpha3-fucosylation of AGP can be used as a putative marker for the development

AGP and its derivatives, prepared by sequential enzymatic cleavage of the carbohydrate units, were tested for their nerve-growth-promoting activities with explants of whole dorsal root ganglia from chick embryos. The results showed that the AGP derivatives with terminal galactose, N-acetylglucosamine, or mannose have marked neurite-promoting

AAT has a molecular weight of *M*r 52 000, and is 12% carbohydrate by weight. The AAT molecule carries a high negative charge because of sialic acid residues on the three complex glycans attached to asparagine residues 46, 83, and 247. Isoelectric focusing of plasma AAT leads to the detection of eight bands, which are numbered M1 to M8 (anodal-low pH to cathodal-high pH). The bands M4 and M6 are the most abundant of the isoforms, making up 40% and 34% of the total plasma AAT,respectively, whereas M3 and M5 are present in only trace amounts (Mills et al., 2001). Fucosylated AAT was analyzed individually and in combination with the currently used marker, alpha-fetoprotein, for the ability to distinguish between a diagnosis of cirrhosis and hepatocellular carcinoma (HCC). The levels of fucosylated AAT were significantly higher in patients with HCC compared to those with cirrhosis (Wang et al., 2009). Remarkably, non-glycosylated AAT showed shorter half life and no ability to interact with IL-8 than compared to glycosylated form of AAT (Bergin et al., 2010). This suggests an importance of AAT glycosylation for its biological activities.

AGP by anti-sLex monoclonal antibody under healthy and disease conditions.

of vascular complications in Type I diabetic patients (Poland et al., 2001).

mediated injury (Williams et al., 1997).

activities (Liu et al., 1988).

**4.1.2 Other APPs**

CRP comprises five identical, non-covalently bound subunits of 206 amino acids (23,017 daltons) arranged in cyclic symmetry (Oliveira et al., 1979). One side of the pentamer participates in binding ligands such as phosphorylcholine, and the other side binds effector molecules such as C1q. When CRP is exposed to denaturing conditions in the presence of a chelating agent, the CRP pentamer is altered to form both individual subunits and aggregates (Gotschlich & Edelman, 1967) designated as modified-CRP (mCRP) (Potempa et al., 1983). The half-life of mCRP in the circulation is <5 min in mice (Motie et al., 1998). These findings indicate that the transport of mCRP from circulation to various sites in the body most likely to be faster than pentameric CRP. mCRP displays antigenic, electrophoretic, and ligand binding reactivities distinct from pentameric CRP (Potempa et al., 1987). Currently has been reported that mCRP is much more potent than pentameric CRP in binding to modified LDL (Singh & Fudenberg, 2009).

Since extravasation and activation of neutrophil granulocytes are essential in the inflammatory response, the effects of CRP on these cells are of particular importance. Stimulation of neutrophils activates a membrane-associated serine protease which leads to the cleavage of biologically active peptides from CRP. CRP peptides 77–82 and 201–206 have been found to inhibit neutrophil chemotaxis to fMLP *in vitro* and to diminish neutrophil influx and protein leakage into alveoli after fMLP induced inflammation in mice (Zouki et al., 1997).

Neutrophil extravasation into inflamed or injured areas involves a complex interaction of leukocytes with endothelial cells *via* regulated expression of surface adhesion molecules. The initial attachment of neutrophils to endothelium is mediated by L-selectin (CD62L) (Díaz-González et al., 1995; Walcheck et al., 1996). L-Selectin is constitutively expressed by

Acute Phase Proteins: Structure and Function Relationship 43

AAT is another example of AAP which can be found *in vivo* in cleaved forms. Cleaved forms of AAT are know to occur when AAT forms an inhibitor complex with serine protease which subsequently dissociates or is degraded, or when it is cleaved by non-target proteases, usually at sites in its reactive loop, without the formation of stable inhibitor complexes. Such cleavage generates a 4 kDa carboxyl-terminal fragment of 36 residues, which remains non-covalently bound to the cleaved AAT. Human cathepsin L, collagenase and stromelysin, and bacterial proteinases from *Staphylococcus aureus*, *Serratia marcescens* metalloproteinase and *Pseudomonas aeruginosa* elastase (Rapala-Kozik et al., 1999) all fall into the latter class and exhibit efficient AAT degrading activity. Recent studies established AAT as a key substrate for gelatinase B (MMP-9) *in vivo* (Liu et al., 2000). It has long been hypothesized that neutrophil elastase-mediated tissue destruction in certain inflammatory diseases such as emphysema, is caused by an imbalance in the ratio of elastase to AAT (Weiss, 1989). The studies of Liu and collaborators provide *in vivo* evidence that this mechanism, mediated by the proteolytic inactivation of AAT by gelatinase B, underlies the pathology of the inflammatory skin disorder called bullous pemphigoid which is initiated by deposit formation at the basement membrane (Jordon et al., 1985). Generated cleaved forms of AAT may contribute to the later phase of polymorphonuclear leukocyte infiltration. Indeed, cleaved AAT was shown to form fibrillar structures and to be a potent

chemoattractant for monocytes (Janciauskiene et al., 1995; Banda et al., 1988).

lupus erythematosus are being studied (Shapira et al., 2011).

**4.3 Oxidized and nitrosylated forms of APPs**

Fragments of AAT have been found in human bile, atherosclerotic plaque, urine and plasma, and have been shown to regulate lipid metabolism, inflammatory cell activation and even to inhibit human HIV-1 expression. It has recently been demonstrated that a specific 20-residue fragment of AAT (C-terminal peptide, residues 377–396, referred to as VIRIP) binds to the gp41 fusion peptide of HIV-1 and prevents the virus from entering target cells, thereby inhibiting HIV-1 infection (Münch et al, 2007). These findings suggest that AAT may play a protective role in HIV-1-infected individuals (Forssmann et al., 2010). We found that the C-terminal fragment of AAT, C-36 peptide, corresponding to residues 359-394 suppresses bile acid synthesis *in vitro* and *in vivo.* The DNA element involved in the C-36-mediated regulation of 7alpha- and 12alpha-hydroxylase promoters mapped to the alpha1-fetoprotein transcription factor site in both promoters. The C-36 peptide prevented binding of FTF to its target DNA recognition site by direct interaction with FTF (Gerbod-Giannone et al., 2002). Hence, the effects of AAT peptides as potential drugs for systemic

Under conditions of compromised oxygen supply, such as occurs in injury, infection or malignancy, oxygen species with free unpaired electrons are generated during mitochondrial electron transport. Referred to as highly reactive oxygen species (ROS), their production causes damage to cell membranes and macromolecules (lipids, proteins and DNA) (Valko et al., 2007). Oxidative changes of protein structure can have a wide range of downstream functional consequences, such as inhibition of enzymatic and binding activities, increased susceptibility to aggregation and proteolysis, increased or decreased uptake by cells, and altered immunogenicity. It is now recognized that oxidation of proteins plays an essential role in the pathogenesis of an important number of degenerative diseases. Compared to controls more oxidized proteins are found in tissues from animals and patients

**4.2.2 AAT**

neutrophils and is released from neutrophils by proteolytic cleavage within minutes after activation with a concomitant upregulation of Mac-1 (CD11b/CD18). The monomeric CRP (but not nCRP) has been found to up-regulate CD11b/CD18 expression and extracellular signal regulated kinase (ERK) activity, suggesting that mCRP may participate in the promotion of neutrophil adhesion to endothelial cells (Zouki et al., 2001). In contrast to CRP, mCRP induces IL-8 secretion in neutrophils (Khreiss et al, 2005) and human coronary artery endothelial cells (Devaraj S et al., 2004), promotes neutrophil-endothelial cell adhesion (Zouki et al., 2001), and delays apoptosis of human neutrophils (Schwedler et al., 2006).

mCRP binds to a number of different ligands to CRP and also exhibits a different set of biologic activities. More recently, it has been shown that mCRP has profound inhibitory effects on tumor growth and metastatic ability of an adenocarcinoma in mice (Kresl et al., 1999). Cross-reactive epitopes of mCRP have also been detected in the fibrous elements of blood vessels and lymphatic organs suggesting that mCRP may be present in extracellular spaces (Samberg et al., 1988). Since mCRP can self-associate into a matrix-like structure (Motie et al., 1996), the naturally occurring antigen may be self-associated aggregates of mCRP, and thus represent a tissue-based as opposed to a blood based form of CRP. mCRP, but not CRP, binds immune complexes (Khreiss et al., 2004), potentiates the activities of activated leukocytes and platelets (Zouki et al., 2001) and stimulates megakaryocyte differentiation in mice (Potempa et al, 1996).

Additionally, both CRP conformations interact differently with components of the complement cascade. M Mihlan et al., 2009, identified for the first time that mCRP, but not pCRP, has a complement-modulating effect. mCRP recruits the complement inhibitor Factor H to the surface of damaged cells or particles, and enhances local complement inhibition both in the fluid phase and on the cellular surface. Thus, by recruiting C1q to the surface of damaged cells, mCRP triggers complement activation resulting in the formation of C3 convertases and C3b surface deposition. However, by binding inhibitor Factor H and enhancing the inhibitory activity, further complement activation, amplification, cytokine release, C9 deposition and terminal membrane attack assembly are inhibited. Furthermore, the phagocytosis of apoptotic particles is increased. This shows how CRP can contribute to an anti-inflammatory scenario and explains how mCRP contributes to the safe removal of damaged apoptotic particles and necrotic cells which may be relevant for diseases such as atherosclerosis (Zipfel& Skerka, 2009).

Using immunofluorescence microscopy (Eisenhardt et al., 2009) have shown the generation of mCRP from CRP on adherent activated platelets, together with the immunohistological colocalization of mCRP with the CD41 antigen in atherosclerotic plaques. These findings suggest that in atherosclerosis mCRP is generated from circulating CRP, and mCRP is then deposited at the atherosclerotic plaque, exerting strong proinflammatory effects (Agrawal et al., 2010).

Despite the growing interest in mCRP, it remains unclear how mCRP is generated and whether it contributes to inflammatory processes such as atherosclerosis. Thus, it appears that both CRP and altered forms of CRP, including mCRP, may each serve important distinct functions in the acute phase and the host defense response to trauma and infectious agents. Therefore, it is important to ascertain the extent of conversion and reversion of CRP to mCRP and possible intermediate forms to help define and understand the biological function(s) of the CRP molecule.

#### **4.2.2 AAT**

42 Acute Phase Proteins – Regulation and Functions of Acute Phase Proteins

neutrophils and is released from neutrophils by proteolytic cleavage within minutes after activation with a concomitant upregulation of Mac-1 (CD11b/CD18). The monomeric CRP (but not nCRP) has been found to up-regulate CD11b/CD18 expression and extracellular signal regulated kinase (ERK) activity, suggesting that mCRP may participate in the promotion of neutrophil adhesion to endothelial cells (Zouki et al., 2001). In contrast to CRP, mCRP induces IL-8 secretion in neutrophils (Khreiss et al, 2005) and human coronary artery endothelial cells (Devaraj S et al., 2004), promotes neutrophil-endothelial cell adhesion (Zouki et al., 2001), and delays apoptosis of human neutrophils (Schwedler et al., 2006). mCRP binds to a number of different ligands to CRP and also exhibits a different set of biologic activities. More recently, it has been shown that mCRP has profound inhibitory effects on tumor growth and metastatic ability of an adenocarcinoma in mice (Kresl et al., 1999). Cross-reactive epitopes of mCRP have also been detected in the fibrous elements of blood vessels and lymphatic organs suggesting that mCRP may be present in extracellular spaces (Samberg et al., 1988). Since mCRP can self-associate into a matrix-like structure (Motie et al., 1996), the naturally occurring antigen may be self-associated aggregates of mCRP, and thus represent a tissue-based as opposed to a blood based form of CRP. mCRP, but not CRP, binds immune complexes (Khreiss et al., 2004), potentiates the activities of activated leukocytes and platelets (Zouki et al., 2001) and stimulates megakaryocyte

Additionally, both CRP conformations interact differently with components of the complement cascade. M Mihlan et al., 2009, identified for the first time that mCRP, but not pCRP, has a complement-modulating effect. mCRP recruits the complement inhibitor Factor H to the surface of damaged cells or particles, and enhances local complement inhibition both in the fluid phase and on the cellular surface. Thus, by recruiting C1q to the surface of damaged cells, mCRP triggers complement activation resulting in the formation of C3 convertases and C3b surface deposition. However, by binding inhibitor Factor H and enhancing the inhibitory activity, further complement activation, amplification, cytokine release, C9 deposition and terminal membrane attack assembly are inhibited. Furthermore, the phagocytosis of apoptotic particles is increased. This shows how CRP can contribute to an anti-inflammatory scenario and explains how mCRP contributes to the safe removal of damaged apoptotic particles and necrotic cells which may be relevant for diseases such as

Using immunofluorescence microscopy (Eisenhardt et al., 2009) have shown the generation of mCRP from CRP on adherent activated platelets, together with the immunohistological colocalization of mCRP with the CD41 antigen in atherosclerotic plaques. These findings suggest that in atherosclerosis mCRP is generated from circulating CRP, and mCRP is then deposited at the atherosclerotic plaque, exerting strong proinflammatory effects (Agrawal et

Despite the growing interest in mCRP, it remains unclear how mCRP is generated and whether it contributes to inflammatory processes such as atherosclerosis. Thus, it appears that both CRP and altered forms of CRP, including mCRP, may each serve important distinct functions in the acute phase and the host defense response to trauma and infectious agents. Therefore, it is important to ascertain the extent of conversion and reversion of CRP to mCRP and possible intermediate forms to help define and understand the biological

differentiation in mice (Potempa et al, 1996).

atherosclerosis (Zipfel& Skerka, 2009).

function(s) of the CRP molecule.

al., 2010).

AAT is another example of AAP which can be found *in vivo* in cleaved forms. Cleaved forms of AAT are know to occur when AAT forms an inhibitor complex with serine protease which subsequently dissociates or is degraded, or when it is cleaved by non-target proteases, usually at sites in its reactive loop, without the formation of stable inhibitor complexes. Such cleavage generates a 4 kDa carboxyl-terminal fragment of 36 residues, which remains non-covalently bound to the cleaved AAT. Human cathepsin L, collagenase and stromelysin, and bacterial proteinases from *Staphylococcus aureus*, *Serratia marcescens* metalloproteinase and *Pseudomonas aeruginosa* elastase (Rapala-Kozik et al., 1999) all fall into the latter class and exhibit efficient AAT degrading activity. Recent studies established AAT as a key substrate for gelatinase B (MMP-9) *in vivo* (Liu et al., 2000). It has long been hypothesized that neutrophil elastase-mediated tissue destruction in certain inflammatory diseases such as emphysema, is caused by an imbalance in the ratio of elastase to AAT (Weiss, 1989). The studies of Liu and collaborators provide *in vivo* evidence that this mechanism, mediated by the proteolytic inactivation of AAT by gelatinase B, underlies the pathology of the inflammatory skin disorder called bullous pemphigoid which is initiated by deposit formation at the basement membrane (Jordon et al., 1985). Generated cleaved forms of AAT may contribute to the later phase of polymorphonuclear leukocyte infiltration. Indeed, cleaved AAT was shown to form fibrillar structures and to be a potent chemoattractant for monocytes (Janciauskiene et al., 1995; Banda et al., 1988).

Fragments of AAT have been found in human bile, atherosclerotic plaque, urine and plasma, and have been shown to regulate lipid metabolism, inflammatory cell activation and even to inhibit human HIV-1 expression. It has recently been demonstrated that a specific 20-residue fragment of AAT (C-terminal peptide, residues 377–396, referred to as VIRIP) binds to the gp41 fusion peptide of HIV-1 and prevents the virus from entering target cells, thereby inhibiting HIV-1 infection (Münch et al, 2007). These findings suggest that AAT may play a protective role in HIV-1-infected individuals (Forssmann et al., 2010). We found that the C-terminal fragment of AAT, C-36 peptide, corresponding to residues 359-394 suppresses bile acid synthesis *in vitro* and *in vivo.* The DNA element involved in the C-36-mediated regulation of 7alpha- and 12alpha-hydroxylase promoters mapped to the alpha1-fetoprotein transcription factor site in both promoters. The C-36 peptide prevented binding of FTF to its target DNA recognition site by direct interaction with FTF (Gerbod-Giannone et al., 2002). Hence, the effects of AAT peptides as potential drugs for systemic lupus erythematosus are being studied (Shapira et al., 2011).

#### **4.3 Oxidized and nitrosylated forms of APPs**

Under conditions of compromised oxygen supply, such as occurs in injury, infection or malignancy, oxygen species with free unpaired electrons are generated during mitochondrial electron transport. Referred to as highly reactive oxygen species (ROS), their production causes damage to cell membranes and macromolecules (lipids, proteins and DNA) (Valko et al., 2007). Oxidative changes of protein structure can have a wide range of downstream functional consequences, such as inhibition of enzymatic and binding activities, increased susceptibility to aggregation and proteolysis, increased or decreased uptake by cells, and altered immunogenicity. It is now recognized that oxidation of proteins plays an essential role in the pathogenesis of an important number of degenerative diseases. Compared to controls more oxidized proteins are found in tissues from animals and patients

Acute Phase Proteins: Structure and Function Relationship 45

and inhibition of cysteine protease. In a study by Ikebe and co-workers (Ikebe et al., 2000) it was suggested that S-NO-AAT exerted a potent cytoprotective effect in liver ischemiareperfusion injury by maintaining the tissue blood flow, inducing hemeoxygenase 1, and suppressing neutrophil-induced liver damage and apoptosis. Interestingly, it was verified that S-NO-AAT expressed similar serine protease inhibitory activity towards pancreatic trypsin and pancreatic and neutrophil elastase as native AAT. Thus, S-NO-AAT may function not only as a simple NO (nitroso) donor but also as a protease inhibitor with a

Function of some APPs is dependent on their complex formation with other molecules and/or on their polymerization. For example, CRP binds to phosphocholine, as well as phosphoethanolamine, microbial surface proteins, chromatin and other ligands (Thompson et al., 1999; Agrawal et al., 2002, Okemefuna et al, 2010). CRP activates the classical pathway of complement by binding to C1q, but its binding to CFH in the alternative pathway has turned out to be more controversial. CRP–ligand interactions lead to the recognition of damaged or apoptotic cells and bacterial pathogens. Ca2+ and phosphocholine bind to the B (binding) face of the pentameric ring, whereas the other A (α-helix) face binds to macromolecular ligands such as C1q. The complex between Hp and Hemoglobin has been studied for decades and represents one of the strongest non-covalent interactions reported in plasma (Nielsen & Moestrup, 2009). Hp also binds apolipoprotein A-I (ApoA-I), and impairs its stimulation of lecithin: cholesterol acyltransferase (LCAT) which plays a major role in reverse cholesterol transport (Cigliano et al., 2009). Hp binds and protects ApoE from oxidative damage (Salvatore et al., 2009). Some APPs are regulated by co-factors which are needed to expose or maintain the functional conformation of the APP. The best example of this phenomenon is antithrombin, which is activated by heparin through induced and transmitted conformational changes that stabilise the proteinase-sensitive active site (Jodan et al., 1987). Another APP, PAI-1, which normally exists in a latent form, can be maintained in its functional form in the presence of plasma vitronectin (Wiman et al., 1988). As well as stabilising PAI-1 in the active conformation, vitronectin also alters the specificity of PAI-1, making it an efficient inhibitor of thrombin. The finding that active PAI-1 specifically inhibits integrin attachment to vitronectin (Stefansson & Lawrence, 1996) further illustrates the unique functional interdependence that exists between PAI-1 and vitronectin. Complexes between APPs and other proteins are also found to be associated with specific diseases. For example, in sera from patients with myeloma and Bence-Jones proteinuria, complexes between AAT and the kappa light chain of immunoglobulins were detected (Laurell et al., 1974). In plasma from diabetic subjects, complexes between AAT and factor Xia, and AAT and heat shock protein-70 (HSP70), as well as glycosylated forms of AAT were detected (Austin et al., 1987; Scott et al., 1998; Finotti et al., 2004). Moreover, complexes between immunoglobulin A and AAT have been detected in the sera and synovial fluid of patients with rheumatoid arthritis, systemic lupus erythematosus and ankylosing spondylitis (Scott et al., 1998). Human tissue kallikrein 3, a serine proteinase commonly known as a prostate-specific antigen (PSA) which correlates with prostate hypertrophy and malignancy, is also known to bind to AAT in sera of subjects with high PSA concentrations (Zhang et al., 2000). Localization of AAT-low-density-lipoprotein (LDL) complexes in atherosclerotic lesions and enhanced degradation of AAT-LDL by macrophages suggested

broad inhibitory spectrum.

**4.4 Complexed and polymerized forms of APPs**

suffering from Alzheimer's disease, rheumatoid arthritis, atherosclerosis or amyotrophic lateral sclerosis (Banfi et al., 2008). For example, in plasma from patients with Alzheimer`s disease the most obviously oxidized proteins were identified as isoforms of AAT and fibrinogen γ-chain precursor proteins. Both these proteins have been suggested to be involved in inflammation processes in Alzheimer's disease (Choi et al., 2002).

*S*-nitrosylation is another important post-transcriptional modification of the APPs and their peptides, which may be involved in NO-dependent signal transductions. For example, a recent study indicates that *S*-nitrosylation can be effectively catalyzed by the copper ion of ceruloplasmin, a major multicopper-containing plasma protein, under physiological conditions (Mani et al., 2004). It is now also conceivable that nitrosylation of thiols is involved in modulation of various biological events, such as functional regulation of receptors, ion channels and synaptic vesicle fusion (Miyamoto et al., 2000).

#### **4.3.1 AAT**

Oxidized AAT has been found in inflammatory exudates at levels of ~5–10% that of total AAT (Wong &Travis, 1988), and AAT recovered from BAL fluid in smokers is 40% less active compared with non-smokers due to oxidation of the P1 methionine (methionine 358 at the active site) to methionine sulfoxide (Carp et al., 1982). The oxidation of AAT by cigarette smoke or free radicals in vivo has been proposed as a mechanism by which elastin and thus alveolar destruction occurs in COPD (Gadek et al., 1979; Beatty et al., 1984). Oxidation of the P1 methionine (methionine 358) or methionine 351 to methionine sulfoxide significantly reduces the ability of AAT to inhibit neutrophil elastase (Taggart et al., 2000). Hydrogen peroxide in cigarette smoke and N-chloroamines and hypochlorous acid in neutrophils can oxidize and inactivate AAT (Ossanna et al., 1986; Scott et al., 1999). Thus the oxidation of AAT by cigarette smoke or free radicals in vivo could lead to a relative deficiency of elastase inhibitors and has been suggested as a mechanism contributing to the development of emphysema and other diseases such as cystic fibrosis, adult respiratory distress syndrome, and bronchiectasis (McGuire et al., 1982; Roum et al., 1993; Izumi-Yoneda et al., 2009). In addition, oxidative inactivation can enhance the susceptibility of AAT to proteolytic attack, particularly by neutrophil elastase and certain bacterial proteases, including thermolysin, aureolysin, serralysin, pseudolysin, *Staphylococcus aureus* serine proteinase, streptopain and periodontain. Thus, oxidation and proteolytic processes in some cases may work synergistically. Moreover, oxidized AAT by itself amplifies and perpetuates the inflammatory processes by directly affecting the functional activities of structural and inflammatory cells or by interacting with other molecules such as IgA and low-density lipoproteins. It has been shown that oxidized AAT significantly induces the production of IL-8 and MCP-1 from a lung epithelial cell line (A549 cells) and in a time- and dosedependent manner and attracts macrophages (Li et al., 2009). Release of oxidants by these inflammatory cells could oxidize newly synthesized AT, which has diffused into the airways and would perpetuate the cycle. This process may be amplified by oxidized AAT induction of MCP-1 synthesis from monocytes (Moraga & Janciauskiene, 2000). These pathways may be one explanation as to why inflammation persists after smoking cessation in chronic obstructive pulmonary disease (Retamales et al., 2001). A complex of oxidized AAT and LDL was isolated from human plasma and was detected in human atherosclerotic lesions of the coronary artery (Donners et al., 2005). The product of AAT nitrosylation, S-NO-AAT, has been shown to have multiple biological functions, including potent anti-microbial activity

suffering from Alzheimer's disease, rheumatoid arthritis, atherosclerosis or amyotrophic lateral sclerosis (Banfi et al., 2008). For example, in plasma from patients with Alzheimer`s disease the most obviously oxidized proteins were identified as isoforms of AAT and fibrinogen γ-chain precursor proteins. Both these proteins have been suggested to be

*S*-nitrosylation is another important post-transcriptional modification of the APPs and their peptides, which may be involved in NO-dependent signal transductions. For example, a recent study indicates that *S*-nitrosylation can be effectively catalyzed by the copper ion of ceruloplasmin, a major multicopper-containing plasma protein, under physiological conditions (Mani et al., 2004). It is now also conceivable that nitrosylation of thiols is involved in modulation of various biological events, such as functional regulation of

Oxidized AAT has been found in inflammatory exudates at levels of ~5–10% that of total AAT (Wong &Travis, 1988), and AAT recovered from BAL fluid in smokers is 40% less active compared with non-smokers due to oxidation of the P1 methionine (methionine 358 at the active site) to methionine sulfoxide (Carp et al., 1982). The oxidation of AAT by cigarette smoke or free radicals in vivo has been proposed as a mechanism by which elastin and thus alveolar destruction occurs in COPD (Gadek et al., 1979; Beatty et al., 1984). Oxidation of the P1 methionine (methionine 358) or methionine 351 to methionine sulfoxide significantly reduces the ability of AAT to inhibit neutrophil elastase (Taggart et al., 2000). Hydrogen peroxide in cigarette smoke and N-chloroamines and hypochlorous acid in neutrophils can oxidize and inactivate AAT (Ossanna et al., 1986; Scott et al., 1999). Thus the oxidation of AAT by cigarette smoke or free radicals in vivo could lead to a relative deficiency of elastase inhibitors and has been suggested as a mechanism contributing to the development of emphysema and other diseases such as cystic fibrosis, adult respiratory distress syndrome, and bronchiectasis (McGuire et al., 1982; Roum et al., 1993; Izumi-Yoneda et al., 2009). In addition, oxidative inactivation can enhance the susceptibility of AAT to proteolytic attack, particularly by neutrophil elastase and certain bacterial proteases, including thermolysin, aureolysin, serralysin, pseudolysin, *Staphylococcus aureus* serine proteinase, streptopain and periodontain. Thus, oxidation and proteolytic processes in some cases may work synergistically. Moreover, oxidized AAT by itself amplifies and perpetuates the inflammatory processes by directly affecting the functional activities of structural and inflammatory cells or by interacting with other molecules such as IgA and low-density lipoproteins. It has been shown that oxidized AAT significantly induces the production of IL-8 and MCP-1 from a lung epithelial cell line (A549 cells) and in a time- and dosedependent manner and attracts macrophages (Li et al., 2009). Release of oxidants by these inflammatory cells could oxidize newly synthesized AT, which has diffused into the airways and would perpetuate the cycle. This process may be amplified by oxidized AAT induction of MCP-1 synthesis from monocytes (Moraga & Janciauskiene, 2000). These pathways may be one explanation as to why inflammation persists after smoking cessation in chronic obstructive pulmonary disease (Retamales et al., 2001). A complex of oxidized AAT and LDL was isolated from human plasma and was detected in human atherosclerotic lesions of the coronary artery (Donners et al., 2005). The product of AAT nitrosylation, S-NO-AAT, has been shown to have multiple biological functions, including potent anti-microbial activity

involved in inflammation processes in Alzheimer's disease (Choi et al., 2002).

receptors, ion channels and synaptic vesicle fusion (Miyamoto et al., 2000).

**4.3.1 AAT**

and inhibition of cysteine protease. In a study by Ikebe and co-workers (Ikebe et al., 2000) it was suggested that S-NO-AAT exerted a potent cytoprotective effect in liver ischemiareperfusion injury by maintaining the tissue blood flow, inducing hemeoxygenase 1, and suppressing neutrophil-induced liver damage and apoptosis. Interestingly, it was verified that S-NO-AAT expressed similar serine protease inhibitory activity towards pancreatic trypsin and pancreatic and neutrophil elastase as native AAT. Thus, S-NO-AAT may function not only as a simple NO (nitroso) donor but also as a protease inhibitor with a broad inhibitory spectrum.

#### **4.4 Complexed and polymerized forms of APPs**

Function of some APPs is dependent on their complex formation with other molecules and/or on their polymerization. For example, CRP binds to phosphocholine, as well as phosphoethanolamine, microbial surface proteins, chromatin and other ligands (Thompson et al., 1999; Agrawal et al., 2002, Okemefuna et al, 2010). CRP activates the classical pathway of complement by binding to C1q, but its binding to CFH in the alternative pathway has turned out to be more controversial. CRP–ligand interactions lead to the recognition of damaged or apoptotic cells and bacterial pathogens. Ca2+ and phosphocholine bind to the B (binding) face of the pentameric ring, whereas the other A (α-helix) face binds to macromolecular ligands such as C1q. The complex between Hp and Hemoglobin has been studied for decades and represents one of the strongest non-covalent interactions reported in plasma (Nielsen & Moestrup, 2009). Hp also binds apolipoprotein A-I (ApoA-I), and impairs its stimulation of lecithin: cholesterol acyltransferase (LCAT) which plays a major role in reverse cholesterol transport (Cigliano et al., 2009). Hp binds and protects ApoE from oxidative damage (Salvatore et al., 2009). Some APPs are regulated by co-factors which are needed to expose or maintain the functional conformation of the APP. The best example of this phenomenon is antithrombin, which is activated by heparin through induced and transmitted conformational changes that stabilise the proteinase-sensitive active site (Jodan et al., 1987). Another APP, PAI-1, which normally exists in a latent form, can be maintained in its functional form in the presence of plasma vitronectin (Wiman et al., 1988). As well as stabilising PAI-1 in the active conformation, vitronectin also alters the specificity of PAI-1, making it an efficient inhibitor of thrombin. The finding that active PAI-1 specifically inhibits integrin attachment to vitronectin (Stefansson & Lawrence, 1996) further illustrates the unique functional interdependence that exists between PAI-1 and vitronectin. Complexes between APPs and other proteins are also found to be associated with specific diseases. For example, in sera from patients with myeloma and Bence-Jones proteinuria, complexes between AAT and the kappa light chain of immunoglobulins were detected (Laurell et al., 1974). In plasma from diabetic subjects, complexes between AAT and factor Xia, and AAT and heat shock protein-70 (HSP70), as well as glycosylated forms of AAT were detected (Austin et al., 1987; Scott et al., 1998; Finotti et al., 2004). Moreover, complexes between immunoglobulin A and AAT have been detected in the sera and synovial fluid of patients with rheumatoid arthritis, systemic lupus erythematosus and ankylosing spondylitis (Scott et al., 1998). Human tissue kallikrein 3, a serine proteinase commonly known as a prostate-specific antigen (PSA) which correlates with prostate hypertrophy and malignancy, is also known to bind to AAT in sera of subjects with high PSA concentrations (Zhang et al., 2000). Localization of AAT-low-density-lipoprotein (LDL) complexes in atherosclerotic lesions and enhanced degradation of AAT-LDL by macrophages suggested

Acute Phase Proteins: Structure and Function Relationship 47

Fig. 4. Schematic diagram showing proposed model for the pathogenesis of emphysema in patients with inherited Z AAT deficiency. Cigarette smoke induces significant oxidation of Z AAT, which accelerates AAT protein polymerization. The plasma deficiency and reduced inhibitory activity of Z AAT would be exacerbated by the oxidation and polymerization of AAT within the lungs, thereby further reducing the antiproteinase screen. This together with the loss of the normal inhibitory effect of AAT on neutrophil chemotaxis and the chemoattractant ability of ΑAT polymers thereby increase tissue damage and accelerate

RM is supported by the Cambridge NIHR Biomedical Research centre. SJ is supported by

Agrawal, A, Elhanan, G & Halper, M. (2010). Dissimilarities in the Logical Modeling of Apparently Similar Concepts in SNOMED CT. *AMIA Annu Symp Proc* 2010, 212-216.

emphysema progression.

**6. Acknowledgment**

**7. References**

Hannover Medical School, Germany.

the involvement of the complex in atherogenesis (Donners et al., 2005). Alpha1 antichymotrypsin/Alzheimer's peptide Abeta (1-42) complexes and ACT polymers have been associated with Alzheimer's disease (Licastro et al., 1997; Sun et al., 2002).Studies of the functional and conformational polymorphism of inhibitory APPs clearly show that some proteins can undergo polymerization due to an inherited mutation, or chemical modification, and obtain new biological activities or reflect undergoing pathological process. A well-characterised example of a mutant APP associated with a disease state is AAT. The most widely studied deficiency variants of AAT are Z and S, which have E342K and E264V mutations respectively (Carrell et al., 1996; Carrell et al., 1892). Polymerisation of these mutants of AAT is known to be involved in AAT deficiency-related diseases such as emphysema, liver cirrhosis, neonatal hepatitis, hepatocellular carcinoma and lung emphysema (Eriksson, 1990) The liver pathology is characterised by the formation of intracellular inclusions of polymerised AAT. Recent studies now indicate that extra-hepatic AAT polymerization may also occur. For example, AAT polymers have been identified in the lungs and circulation of Z AAT deficiency subjects. Importantly, like other modified forms, AAT polymers lack protease inhibitor activity which will exaggerate the severe deficiency, but also exhibit additional biological functions that may be relevant in pathological processes (Mahadeva et al., 2005; Mulgrew et al., 2004) (Figure 5). The susceptibility of Z AAT individuals who smoke to develop chronic obstructive pulmonary disease is in part related to the combination of the severe anti-elastase deficiency arising from an absolute and functional reduction in neutrophil elastase inhibitory capacity (Stoller & Aboussouan, 2005), and the independent multiple effects of cigarette smoke on inflammatory cells and molecules (Barnes et al., 2003). Our current data clearly demonstrate that cigarette smoke promotes polymerization of Z mutant AAT, but not of the normal, M variant of AAT. Thus cigarette smoke directly accelerates polymerization of Z AAT *via*  oxidation of the protein leading to further depletion of the neutrophil protease protection in the lung and enhanced neutrophil influx (Alam et al., 2010). The data uniquely suggests that rather than the major risk factors for chronic obstructive pulmonary disease namely cigarette smoke and Z AAT deficiency having independent additive effects, they directly interact to create an effect greater than the sum of the individual risks.

#### **5. General conclusions**

Inflammation is a complex, highly orchestrated process involving many cell types and molecules, some of which initiate, amplify, or sustain the process, some of which attenuate it, and some of which aid resolution. A number of the participating AAPs are multifunctional and contribute to both the enhancement and the inhibition of inflammation at different points in its evolution. The outcome of the acute inflammatory response is most likely to be determined by the orchestrated generation of a specific profile of APPs, their concentrations and molecular forms in the microenvironment. Diseases associated with chronic inflammation may be due to an inadequate acute-phase response driven by APPs, their concentration and molecular form and/or an inability to eliminate invading pathogens and to rapidly to resolve the inflammatory processes. Often, a single APP is regarded as a marker for inflammation to aid in diagnosis and assesses response to treatement, however we believe one needs to see the profile of several APPs in action to understand function in relation to disease which may help in turn to determine prognosis.

the involvement of the complex in atherogenesis (Donners et al., 2005). Alpha1 antichymotrypsin/Alzheimer's peptide Abeta (1-42) complexes and ACT polymers have been associated with Alzheimer's disease (Licastro et al., 1997; Sun et al., 2002).Studies of the functional and conformational polymorphism of inhibitory APPs clearly show that some proteins can undergo polymerization due to an inherited mutation, or chemical modification, and obtain new biological activities or reflect undergoing pathological process. A well-characterised example of a mutant APP associated with a disease state is AAT. The most widely studied deficiency variants of AAT are Z and S, which have E342K and E264V mutations respectively (Carrell et al., 1996; Carrell et al., 1892). Polymerisation of these mutants of AAT is known to be involved in AAT deficiency-related diseases such as emphysema, liver cirrhosis, neonatal hepatitis, hepatocellular carcinoma and lung emphysema (Eriksson, 1990) The liver pathology is characterised by the formation of intracellular inclusions of polymerised AAT. Recent studies now indicate that extra-hepatic AAT polymerization may also occur. For example, AAT polymers have been identified in the lungs and circulation of Z AAT deficiency subjects. Importantly, like other modified forms, AAT polymers lack protease inhibitor activity which will exaggerate the severe deficiency, but also exhibit additional biological functions that may be relevant in pathological processes (Mahadeva et al., 2005; Mulgrew et al., 2004) (Figure 5). The susceptibility of Z AAT individuals who smoke to develop chronic obstructive pulmonary disease is in part related to the combination of the severe anti-elastase deficiency arising from an absolute and functional reduction in neutrophil elastase inhibitory capacity (Stoller & Aboussouan, 2005), and the independent multiple effects of cigarette smoke on inflammatory cells and molecules (Barnes et al., 2003). Our current data clearly demonstrate that cigarette smoke promotes polymerization of Z mutant AAT, but not of the normal, M variant of AAT. Thus cigarette smoke directly accelerates polymerization of Z AAT *via*  oxidation of the protein leading to further depletion of the neutrophil protease protection in the lung and enhanced neutrophil influx (Alam et al., 2010). The data uniquely suggests that rather than the major risk factors for chronic obstructive pulmonary disease namely cigarette smoke and Z AAT deficiency having independent additive effects, they directly

interact to create an effect greater than the sum of the individual risks.

Inflammation is a complex, highly orchestrated process involving many cell types and molecules, some of which initiate, amplify, or sustain the process, some of which attenuate it, and some of which aid resolution. A number of the participating AAPs are multifunctional and contribute to both the enhancement and the inhibition of inflammation at different points in its evolution. The outcome of the acute inflammatory response is most likely to be determined by the orchestrated generation of a specific profile of APPs, their concentrations and molecular forms in the microenvironment. Diseases associated with chronic inflammation may be due to an inadequate acute-phase response driven by APPs, their concentration and molecular form and/or an inability to eliminate invading pathogens and to rapidly to resolve the inflammatory processes. Often, a single APP is regarded as a marker for inflammation to aid in diagnosis and assesses response to treatement, however we believe one needs to see the profile of several APPs in action to understand function in relation to disease which may help

**5. General conclusions**

in turn to determine prognosis.

Fig. 4. Schematic diagram showing proposed model for the pathogenesis of emphysema in patients with inherited Z AAT deficiency. Cigarette smoke induces significant oxidation of Z AAT, which accelerates AAT protein polymerization. The plasma deficiency and reduced inhibitory activity of Z AAT would be exacerbated by the oxidation and polymerization of AAT within the lungs, thereby further reducing the antiproteinase screen. This together with the loss of the normal inhibitory effect of AAT on neutrophil chemotaxis and the chemoattractant ability of ΑAT polymers thereby increase tissue damage and accelerate emphysema progression.

#### **6. Acknowledgment**

RM is supported by the Cambridge NIHR Biomedical Research centre. SJ is supported by Hannover Medical School, Germany.

#### **7. References**

Agrawal, A, Elhanan, G & Halper, M. (2010). Dissimilarities in the Logical Modeling of Apparently Similar Concepts in SNOMED CT. *AMIA Annu Symp Proc* 2010, 212-216.

Acute Phase Proteins: Structure and Function Relationship 49

Carp, H, et al. (1982). Potential mechanism of emphysema: alpha 1-proteinase inhibitor

Carrell, RW, et al. (1996). Alpha 1-antitrypsin deficiency. A conformational disease. *Chest*

Casey, R, et al. (2008). The acute-phase reactant C-reactive protein binds to

Cermak, J, et al. (1993). Cell heme uptake induces ferritin synthesis resulting in altered

Chapman, KR, Stockley, RA, Dawkins, C, Wilkes, MM, & Navickis, RJ. (2009).

Cheng, N, et al. (2008). Cutting edge: TLR2 is a functional receptor for acute-phase serum

Choi, J, et al. (2002). Identification of oxidized plasma proteins in Alzheimer's disease.

Chowdhury, MM, et al. (2002). Severe panniculitis caused by homozygous ZZ alpha1-

Churg, A, et al. (2001). Alpha-1-antitrypsin and a broad spectrum metalloprotease inhibitor, RS113456, have similar acute anti-inflammatory effects*. Lab Inves* 81, 1119-1131. Cid, MC, et al. (1993). Identification of haptoglobin as an angiogenic factor in sera from

Cigliano, L, et al. (2009). Haptoglobin binds the antiatherogenic protein apolipoprotein E-

Connolly, M, et al. (2010). Acute serum amyloid A induces migration, angiogenesis, and

Costello M, Fiedel BA & Gewurz H. (1979). Inhibition of platelet aggregation by native and

Costello, MJ, Gewurz, H & Siegel, JN. (1984). Inhibition of neutrophil activation by alpha1-

Culley, FJ, et al. (1996). C-reactive protein binds to a novel ligand on Leishmania donovani and increases uptake into human macrophages. *J Immunol* 156, 4691-4696. Daemen, MA, et al. (2000). Functional protection by acute phase proteins alpha(1)-acid

De Graaf, TW, et al. (1993). Inflammation-induced expression of sialyl Lewis X-containing

preventing apoptosis and inflammation. *Circulation* 102, 1420-1426.

Carrell, RW, & Lomas, DA. (1997). Conformational disease. *Lancet 350*, 134-138.

human phagocytes. *Infect Immun* 76, 1298-1304.

*J Clin Invest* 78, 1427-1431.

amyloid A. *J Immunol.* 181, 22-26.

*Br J Dermatol* 147, 1258-1261.

*Biochem Biophys Res Commun* 293, 1566-1570.

patients with systemic vasculitis. *J Clin Invest* 91, 977-985.

desialised alpha-1 acid glycoprotein. *Nature* 281, 677-678.

mouse chimera model*. J Immunol* 184, 6427-6437.

acid glycoprotein. *Clin Exp Immunol* 55, 465-472.

110, 243S-247S.

5308-5313.

177-184.

6171.

*Med* 177, 657-666.

recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capacity. *Proc Natl Acad Sci U S A* 79, 2041-2045. Carrell , R. (1986). alpha 1-Antitrypsin: molecular pathology, leukocytes, and tissue damage*.*

phosphorylcholine-expressing Neisseria meningitidis and increases uptake by

oxidant sensitivity: possible role in chemotherapy efficacy. *Tumor Cancer Res* 53,

Augmentation therapy for alpha1 antitrypsin deficiency: a meta-analysis. *COPD* 6,

antitrypsin deficiency treated successfully with human purified enzyme (Prolastin).

impairment of apolipoprotein E stimulation of both lecithin:cholesterol acyltransferase activity and cholesterol uptake by hepatocytes*. FEBS J* 276, 6158-

inflammation in synovial cells in vitro and in a human rheumatoid arthritis/SCID

glycoprotein and alpha(1)-antitrypsin against ischemia/reperfusion injury by

glycan structures on alpha 1-acid glycoprotein (orosomucoid) in human sera. *J Exp*

Alam, S, Li Z, Janciauskiene, S, & Mahadeva, R. (2010). Oxidation of Z {alpha}1-antitrypsin

Al-Omari, M, et al. (2011). The acute phase protein, alpha1-antitrypsin, inhibits neutrophil calpain I and induces random migration. *Mol Med* Apr 11 [Epub ahead of print]. Anderson, N, et al. (2002). A preliminary evaluation of the differences in the glycosylation of

Arredouani, M, et al. (2003). Haptoglobin directly affects T cells and suppresses T helper cell

Assicot, M, et al. (1993). High serum procalcitonin concentrations in patients with sepsis and

Austin, GE, Mullins, RH & Morin, LG. (1987). Non-enzymic glycation of individual plasma proteins in normoglycemic and hyperglycemic patients*. Clin Chem* 33, 2220-2224. Ballou, SP& Lozanski G. (1992). Induction of inflammatory cytokine release from cultured

Banda, MJ, Rice, AG, Griffin, GL, & Senior, RM. (1988). Alpha 1-proteinase inhibitor is a

Banfi, C, et al. (2008). Oxidized proteins in plasma of patients with heart failure: role in

Barnes, PJ. (2003). New treatments for chronic obstructive pulmonary disease. *Ann Ist Super*

Beatty, K, Matheson, N, & Travis, J. (1984). Kinetic and chemical evidence for the inability of

Berman, S, Gewurz, H, & Mold, C. (1986). Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis. *J Immunol* 136, 1354-1359. Bisoendial, R, et al. (2005). In vivo effects of C-reactive protein (CRP)-infusion into humans.

Blackburn, WD. (1994). Validity of acute phase proteins as markers of disease activity. *J*

Blanco, I, et al. (2006). Estimated numbers and prevalence of PI\*S and PI\*Z alleles of alpha1 antitrypsin deficiency in European countries. *Eur Respir J* 27, 77-84.

Boutten, A, et al. (1992). Alpha 1-acid glycoprotein potentiates lipopolysaccharide-induced

Brand, K, et al. (1996). Activated transcription factor nuclear factor-kappa B is present in the

Brinckerhoff, CE, et al. (1989). Autocrine induction of collagenase by serum amyloid A-like

Bucurenci, N, et al. (1992). Inhibition of neutrophil superoxide production by human plasma

secretion of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha by human monocytes and alveolar and peritoneal macrophages. *Eur J Immunol* 22,

Berg, RD. (1996). The indigenous gastrointestinal microflora.*Trends Microbiol* 4, 430-435. Bergin, DA, et al. (2010). α-1 Antitrypsin regulates human neutrophil chemotaxis induced by

soluble immune complexes and IL-8*. J Clin Invest* 120, 4236-4250.

neutrophil chemoattractant after proteolytic inactivation by macrophage elastase. *J*

oxidized alpha 1-proteinase inhibitor to protect lung elastin from elastolytic

Emphysema. *Am J Respir Cell Mol Biol Epub ahead of print*.

human monocytes by C-reactive protein. *Cytokine* 4, 361-368

Barter ,PJ, et al. (2004). Antiinflammatory properties of HDL*. Circ Res* 95, 764-772.

degradation. *Hoppe Seylers Z Physiol Chem* 365, 731-736.

atherosclerotic lesion. *J Clin Invest* 97, 1715-1722.

alpha 1-antitrypsin. *FEBS Lett* 300, 21-24.

and beta 2-microglobulin-like proteins. *Science* 243, 655-7.

type 2 cytokine release. *Immunology* 108, 144-151.

endothelial damage. *Eur J Heart Fail* 10, 244-251.

infection. *Lancet* 341, 515-518.

*Biol Chem* 263, 4481-4484.

*Sanita* 39, 573-582.

*Circ Res* 97, e115-116.

*Rheumatol* 42, 9–13.

2687-2695.

365-372.

by Cigarette Smoke Induces Polymerization: A Novel Mechanism of Early-onset

alpha-1-acid glycoprotein between individual liver diseases. *Biomed Chromatogr* 16,

Acute Phase Proteins: Structure and Function Relationship 51

Eriksen, N, & Benditt, EP. (1980). Isolation and characterization of the amyloid-related

Finotti, P, & Pagetta, A. (2004). A heat shock protein70 fusion protein with alpha1-

Fournier, T, Medjoubi-N, N & Porquet, D. (2000). Alpha-1-acid glycoprotein. *Biochim Biophys*

Forney, JR, Yang, S, & Healey, MC. (1996). Interaction of the human serine protease inhibitor alpha-1-antitrypsin with Cryptosporidium parvum. *J Parasitol* 82, 496-502. Fröhlander, N, Ljunggren, O, & Lerner, UH. (1991). Haptoglobin synergistically potentiates

Gabay, C, & Kushner, I. (1999). Acute-phase proteins and other systemic responses to

Gadek, JE, Fells, GA, & Crystal, RG. (1979). Cigarette smoking induces functional

Gerbod-Giannone, MC, et al. (2002). Suppression of cholesterol 7alpha-hydroxylase

Gershov, D, et al. (2000). C-Reactive protein binds to apoptotic cells, protects the cells from

Gettins, PG. (2002). Serpin structure, mechanism, and function. *Chem Rev* 102, 4751-4804. Graziadei, I, et al. (1997). Unidirectional upregulation of the synthesis of the major iron

Gitlin, JD, Gitlin, JI, & Gitlin, D. (1977). Localizing of C-reactive protein in synovium of

Griese, M, et al. (2007). alpha1-Antitrypsin inhalation reduces airway inflammation in cystic

Gooptu, B, & Lomas, DA. (2009). Conformational pathology of the serpins: themes, variations, and therapeutic strategies. *Annu Rev Biochem* 78, 147-176. Gross, B, et al. (2009). New Findings in PiZZ alpha1-antitrypsin deficiency-related

Gruys, E, Obwolo, MJ, & Toussaint, MJ. (1994) Diagnostic significance of the major acute phase proteins in veterinary clinical chemistry: a review. *Vet Bull* 64, 1009–1018. Gotschlich, EC & Edelman, GM. (1967). Binding properties and specificity of C-reactive

Gutsmann, T, et al. (2001). Dual role of lipopolysaccharide (LPS)-binding protein in

Hack, CE, et al. (1997). Role for secretory phospholipase A2 and C-reactive protein in the

patients with rheumatoid arthritis. *Arthritis Rheum* 20, 1491-1499.

intravenous augmentation therapy. *Dermatology* 218, 370-375.

osteoblasts. *Biochem Biophys Res Commun* 178, 343-351.

inflammation. *N Engl J Med* 340: 448-454.

autoimmunity. *J Exp Med* 192, 1353-1364.

fibrosis patients. *J Eur Respir* 29, 240-250.

protein. *Proc Natl Acad Sci U S A* 57, 706-712.

removal of injured cells. *Immunol Today* 18, 111-115.

cells. *Infect Immun* 69, 6942-6950.

protein alpha1-antitrypsin*. J Hepatol* 27, 716-725.

77, 6860-6864.

*Acta* 1482, 157-171.

297-305.

1316.

42980.

apoprotein (SAA) from human high density lipoprotein. *Proc Natl Acad Sci U S A*

antitrypsin in plasma of type 1 diabetic subjects. *Biochem Biophys Res Commun* 315,

bradykinin and thrombin induced prostaglandin biosynthesis in isolated

antiprotease deficiency in the lower respiratory tract of humans. *Science* 206, 1315-

transcription and bile acid synthesis by an alpha1-antitrypsin peptide via interaction with alpha1-fetoprotein transcription factor. *J Biol Chem* 277, 42973-

assembly of the terminal complement components, and sustains an antiinflammatory innate immune response: implications for systemic

proteins, transferrin-receptor and ferritin, in HepG2 cells by the acute-phase

panniculitis.Demonstration of skin polymers and high dosing requirements of

neutralization of LPS and enhancement of LPS-induced activation of mononuclear

Dehghan, A, et al. (2007). Risk of type 2 diabetes attributable to C-reactive protein and other

De Martin, R, et al. (2000). The transcription factor NF-kappa B and the regulation of

Devaraj, S, Kumaresan, PR & Jialal, I. (2004). Effect of C-reactive protein on chemokine expression in human aortic endothelial cells. *J Mol Cell Cardiol* 36, 405-410. De Santo, C, et al. (2010). Invariant NKT cells modulate the suppressive activity of IL-10-

Díaz-González, F, et al. (1995). Prevention of in vitro neutrophil-endothelial attachment

Dinarello, CA. (1983). Pathogenesis of fever during hemodialysis. *Contrib Nephrol* 36, 90–99. Donners, MM, et al. (2005). Proteomic analysis of differential protein expression in human

Dowd, SK, Rodgers, GC, & Callen, JP. (1995). Effective treatment with alpha 1-protease

Drenth JP, van der Meer JW & Kushner I. (1996). Unstimulated peripheral blood

Du Clos, TW. (1989). C-reactive protein reacts with the U1 small nuclear ribonucleoprotein. *J*

Du Clos, TW, Zlock, LT, & Rubin, RL. (1988). Analysis of the binding of C-reactive protein to

Dupre, T, et al. (2001). Congenital disorder of glycosylation Ia with deficient

Eckhardt ER, et al. (2010). Intestinal epithelial serum amyloid A modulates bacterial growth

Edwards, DH, et al. (1986). Haptoglobin-haemoglobin complex in human plasma inhibits

Eisenhardt, SU, Habersberger, J & Peter, K. (2009). Monomeric C-reactive protein generation

Elliott, MA, et al. (1997). Investigation into the concanavalin A reactivity, fucosylation and

Engström, G, et al. (2004). Incidence of fatal or repaired abdominal aortic aneurysm in

phosphomannomutase activity but normal plasma glycoprotein pattern. *Clin Chem*

in vitro and pro inflammatory responses in mouse experimental colitis. *BMC*

endothelium dependent relaxation: evidence that endothelium derived relaxing

on activated platelets: the missing link between inflammation and

oligosaccharide microheterogeneity of alpha 1-acid glycoprotein expressed in the sera of patients with rheumatoidarthritis. *J Chromatogr B Biomed Sci Appl* 688, 229-

relation to inflammation-sensitive plasma proteins. *Arterioscler Thromb Vasc Biol* 24,

secreting neutrophils differentiated with serum amyloid A. *Nat Immunol* 11, 1039-

through shedding of L-selectin by nonsteroidal antiinflammatory drugs. *J Clin*

inhibitor of chronic cutaneous vasculitis associated with alpha 1-antitrypsin

mononuclear cells from patients with the hyper-IgD syndrome produce cytokines capable of potent induction of C-reactive protein and serum amyloid A in Hep3B

vascular cell function. *Arterioscler Thromb Vasc Biol* 20, E83-88.

atherosclerotic plaque progression. *J Pathol 206*, 39-45.

Du Clos, TW. (2000). Function of C-reactive protein. *Ann Med* 32, 274-278.

Eaton, JW, et al. (1982). Haptoglobin: a natural bacteriostat. *Science* 215, 691-693.

factor acts as a local autocoid. *Cardiovasc Res* 20, 549-556.

atherothrombotic risk. *Trends Cardiovasc Med* 19, 232-237.

histones and chromatin. *J Immunol* 141, 4266-4270.

deficiency. *J Am Acad Dermatol* 33, 913-916.

cells. *J Immunol* 157, 400-404.

*Immunol* 143, 2553-2559.

47, 132-134.

237.

337-341

*Gastroenterol* 10, 133.

risk factors. *Diabetes Care* 30, 2695-2699.

1046.

*Invest* 95, 1756-1765.

Acute Phase Proteins: Structure and Function Relationship 53

Kalis, M, et al. (2010). α 1-antitrypsin enhances insulin secretion and prevents cytokine-

Kakehi, K, Kinoshita, M & Nakano, M. (2002). Analysis of glycoproteins and the

Kaźmierczak, M, et al. (1995). Changes of acute phase proteins glycosylation profile as a possible prognostic marker in myocardial infarction. *Int J Cardiol* 49, 201-207. Kew, RR, Hyers, TM, & Webster, RO. (1990). Human C-reactive protein inhibits neutrophil

Khreiss, T, et al. (2004). Opposing effects of C-reactive protein isoforms on shear-induced

Khreiss, T, et al. (2005). Loss of pentameric symmetry inC-reactive protein induces

Knappstein, S, et al. (2004). Alpha 1-antitrypsin binds to and interferes with functionality of

Koenig, W, et al. (1999). C-Reactive protein, a sensitive marker of inflammation, predicts

Koulmanda, M, et al. (2008). Curative and beta cell regenerative effects of alpha1-antitrypsin

Koj , A. (1985) Cytokines regulating acute inflammation and synthesis of acute phase

Kresl, JJ, Potempa, LA, Anderson, B & Radosevich, JA.(1999). Inhibition of mouse mammary

Kueppers, F. (2011). The role of augmentation therapy in alpha-1 antitrypsin deficiency.

Kurosky, A, et al. (1980). Covalent structure of human haptoglobin: a serine protease

Kushner, I. (1982). The phenomenon of the acute phase response. *Ann N Y Acad Sci* 389, 39-

Kushner, I & Feldmann, G. (1978). Control of the acute phase response. Demonstration of C-

Kushner, I, Gewurz, H, & Benson, MD. (1981). C-reactive protein and the acute-phase

reactive protein synthesis and secretion by hepatocytes during acute inflammation

Disease) Augsburg Cohort Study, 1984 to 1992. *Circulation* 99, 237-242. Koenig, W, et al. (2006). Increased concentrations of C-reactive protein and IL-6 but not IL-

2002. *Arterioscler Thromb Vasc Biol* 26, 2745-2751.

homolog. *Proc Natl Acad Sci U S A* 77, 3388-3392.

oligosaccharides thereof by high-performance capillary electrophoresis-significance in regulatory studies on biopharmaceutical products. *Biomed Chromatogr* 16, 103-

chemotaxis in vitro: possible implications for the adult respiratory distress

neutrophil-platelet adhesion and neutrophil aggregation in whole blood.

interleukin-8 secretion through peroxynitrite signaling in human neutrophils. *Circ*

EspB from atypical and typical enteropathogenic Escherichia coli strains. *Infect*

future risk of coronary heart disease in initially healthy middle-aged men: results from the MONICA (Monitoring Trends and Determinants in Cardiovascular

18 are independently associated with incident coronary events in middle-aged men and women: results from the MONICA/KORA Augsburg case-cohort study, 1984-

treatment in autoimmune diabetic NOD mice*. Proc Natl Acad Sci U S A* 105, 16242-

adenocarcinoma (EMT6) growth and metastases in mice by a modified form of C-

mediated apoptosis in pancreatic β-cells. *Islets* 2, 185-189.

syndrome. *J Lab Clin Med* 115, 339-345.

*Circulation* 110, 2713-2720.

*Res* 97, 690-697.

16247.

48.

*Immun* 72, 4344-4350.

proteins. *Blut* 51, 267-274.

reactive protein. *Tumour Biol* 20, 72-87.

*Curr Med Res Opin* 27, 579-588.

in the rabbit. *J Exp Med* 148, 466-77.

response. *J Lab Clin Med* 97, 739-749.

115.

Hanriot, D, et al. (2008). C-reactive protein induces pro- and anti-inflammatory effects,

Hattori, Y, Matsumura, M, Kasai, K. (2003). Vascular smooth muscle cell activation by C-

Havenaar, EC, et al. (1998). Severe rheumatoid arthritis prohibits the pregnancy-induced

Haverkate, F, et al. (1997). Production of C-reactive protein and risk of coronary events in

He, R, Sang, H & Ye, RD. (2003). Serum amyloid A induces IL-8 secretion through a G

Heuertz, RM, et al. (1999). C-reactive protein inhibits chemotactic peptide-induced p38

Hochepied, T, et al. (2003). Alpha(1)-acid glycoprotein: an acute phase protein with

Hrycaj, P, et al. (1996). Microheterogeneity of acute-phase glycoproteins in patients with

Ianni, M, et al. (2010). Altered glycosylation profile of purified plasma ACT from

Ikebe, N, et al. (2000). Protective effect of S-nitrosylated alpha(1)-protease inhibitor on hepatic ischemia-reperfusion injury. *J Pharmacol Exp Ther* 295, 904-911. Ingenbleek, Y, & Bernstein L. (1999). The stressful condition as a nutritionally dependent

Izumi-Yoneda, N, et al. (2009). Alpha-1 antitrypsin activity is decreased in human amnion in premature rupture of the fetal membranes. *Mol Hum Reprod* 15, 49-57. Jahangiri, A, et al. (2009). HDL remodeling during the acute phase response. *Arterioscler*

Janciauskiene, S. (2001). Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles. *Biochim Biophys Acta* 1535, 221-235. Janciauskiene, S, Carlemalm, E, & Eriksson, S. (1995). In vitro fibril formation from alpha1 antitrypsin-derived C-terminal peptides. *Biol Chem Hoppe Seyler* 376, 415-423. Janciauskiene, SM, Nita, IM, & Stevens, T. (2007). Alpha1-antitrypsin, old dog, new tricks.

Janciauskiene, S, et al. (2008). Alpha1-antitrypsin inhibits the activity of the matriptase

Janciauskiene, SM, et al. (2011). The discovery of α1-antitrypsin and its role in health and

Jie, Z, et al. (2003). Protective effects of alpha 1-antitrypsin on acute lung injury in rabbits

Jones, SA, et al. (1999). C-reactive protein: a physiological activator of interleukin 6 receptor

Jordon, RE, Kawana, S, & Fritz, KA. (1985). Immunopathologic mechanisms in pemphigus

catalytic domain in vitro. *Am J Respir Cell Mol Biol* 39, 631-637.

Alpha1-antitrypsin exerts in vitro anti-inflammatory activity in human monocytes

Disabilities Angina Pectoris Study Group. *Lancet* 349, 462-466.

protein-coupled receptor, FPRL1/LXA4R. *Blood* 101, 1572-1581.

*Haemost.* 99, 558-569.

*Chem* 274, 17968-17974.

25-34.

reactive protein. *Cardiovasc Res* 58, 186-195.

pulmonary sarcoidosis. *Eur Respir J* 9, 313-318

Alzheimer's disease. *Immun Ageing 7* Suppl, S6.

adaptive dichotomy. *Nutrition* 15, 305–320.

by elevating cAMP. *J Biol Chem* 282, 8573-8582.

Javid, J. (1978). Human haptoglobins. *Curr Top Hematol* 1, 151-192.

induced by endotoxin. *Chin Med J (Engl)* 116, 1678-1682.

and bullous pemphigoid. *J Invest Dermatol* 85, 72s-78s.

disease. *Respir Med*. *[Epub ahead of print]*.

shedding. *J Exp Med* 189, 599-604.

*Thromb Vasc Biol* 29, 261-267.

including activation of the liver X receptor alpha, on human monocytes. *Thromb*

decrease in alpha3-fucosylation of alpha1-acid glycoprotein. *Glycoconj J* 15, 723-729.

stable and unstable angina. European Concerted Action on Thrombosis and

mitogen-activated protein kinase activity and human neutrophil movement. *J Biol*

inflammatory and immunomodulating properties. *Cytokine Growth Factor Rev* 14,

Acute Phase Proteins: Structure and Function Relationship 55

Lysenko, E, et al. (2000). The position of phosphorylcholine on the lipopolysaccharide of

Lögdberg, L & Wester, L. (2000). Immunocalins: a lipocalin subfamily that modulates immune and inflammatory responses. *Biochim Biophys Acta* 1482, 284-297. Mackiewicz, A, et al. (1987). Monokines regulate glycosylation of acute-phase proteins. *J Exp*

Maes, OC, et al. (2006). Characterization of alpha1-antitrypsin as a heme oxygenase-1

Mahadeva, R, et al., (2005) Polymers of Z alpha1-antitrypsin co-localize with neutrophils in emphysematous alveoli and are chemotactic in vivo. *Am J Pathol* 166, 377-386 Malle, E, & De Beer, FC. (1996). Human serum amyloid A (SAA) protein: a prominent acute-

Mani, K, et al. (2004). Involvement of glycosylphosphatidylinositol-linked ceruloplasmin in

Marumo, T, et al. (1997). Platelet-derived growthfactor-stimulated superoxide anion

McGuire, WW, et al. (1982). Studies on the pathogenesis of the adult respiratory distress

Mills, K, et al. (2001). Identification of alpha(1)-antitrypsin variants in plasma with the use of

Miyamoto, Y, Akaike, T, & Maeda, H. (2000). S-nitrosylated human alpha(1)-protease

Mold, C, Gewurz, H, & Du Clos, TW. (1999). Regulation of complement activation by C-

Mold, C, Kingzette, M, & Gewurz, H. (1984). C-reactive protein inhibits pneumococcal

Mold, C, et al. (1981). C-reactive protein is protective against Streptococcus pneumoniae

Mold, C, et al. (2002). C-reactive protein mediates protection from lipopolysaccharide through interactions with Fc gamma R. *J Immunol Today* 169, 7019-7025. Moore, DF, et al. (1997). Alpha-1-acid (AAG,orosomucoid) glycoprotein: interaction with bacterial lipopolysaccharide and protection from sepsis. *Inflammation* 21, 69-82. Moraga, F, & Janciauskiene S. (2000). Activation of primary human monocytes by the

Moshage, HJ, et al. (1987). Study of the molecular mechanism of decreased liver synthesis of

Motie, M, Schaul, KW & Potempa, LA. (1998). Biodistribution and clearance of 125I-labeled

Muchitsch, EM, Auer, W & Pichler, L. (1998). Effects of alpha 1-acid glycoprotein in different

Mulgrew, AT, et al., (2004) Z alpha1-antitrypsin polymers in the lung and acts as a

C-reactive protein and 125I-labeled modified C-reactive protein in CD-1 mice. *Drug*

oxidized form of alpha1-antitrypsin. *J Biol Chem* 275, 7693-7700.

rodent models of shock. *Fundam Clin Pharmacol.* 12, 173-181.

albumin in inflammation. *J Clin Invest* 79, 1635-1641.

neutrophil chemoattractant. *Chest*, 125, 1952-1957.

activation of the alternative pathway by increasing the interaction between factor H

the copper/zinc-nitric oxide-dependent degradation of glypican-1 heparan sulfate

production modulates activation of transcription factor NF-kappaB and expression of monocyte chemoattractant protein1 in human aortic smooth muscle cells.

suppressor in Alzheimer plasma. *Neurobiol Dis* 24, 89-100.

in rat C6 glioma cells. *J Biol Chem* 279, 12918-12923

phase reactant for clinical practice*. Eur J Clin Invest* 26, 427-435.

mediated killing. *Mol Microbiol* 35, 234-245.

*Med* 166, 253-258.

*Circulation* 96, 2361-2367.

syndrome*. J Clin Invest* 69, 543-553.

and C3b. *J Immunol* 133, 882-885.

*Metab Dispos* 26, 977-981.

infection in mice. *J Exp Med* 54, 1703-8.

proteomic technology. *Clin Chem* 47, 2012-22.

reactive protein. *Immunopharmacology* 42, 23-30.

inhibitor. *Biochim Biophys Acta* 1477, 90-97.

Haemophilus influenzae affects binding and sensitivity to C-reactive protein-

Kushner, I, & Mackiewicz, A. (1993). The acute-phase response: an overview. In: *Acute Phase*

Laine, A, et al. (1990). Comparison of the effects of purified human alpha 1-

1-proteinase inhibitor inhibits natural killing. *Clin Chim Acta* 190, 163-173. Laurell, CB, & Thulin, E. (1974). Complexes in plasma between light chain kappa

Lerner, UH, & Fröhlander, N. (1992). Haptoglobin-stimulated bone resorption in neonatal

Lewis, EC, et al. (2008). Alpha1-Antitrypsin monotherapy induces immune tolerance during islet allograft transplantation in mice. *Proc Natl Acad Sci U S A* 105, 16236-16241. Liang, YJ, et al. (2006). C-reactive protein activates the nuclear factor-kappaB pathway and

Libert, C, Brouckaert, P & Fiers, W. (1994). Protection by alpha 1-acid glycoprotein against

Libert, C, et al. (1996). alpha1-Antitrypsin inhibits the lethal response to TNF in mice. *J*

Li, Z, et al. (2009). Oxidized{alpha}1-antitrypsin stimulates the release of monocyte

Licastro, F, et al. (1997). Monomeric and polymeric forms of alpha-1 antichymotrypsin in

Lieberman J. (2000). Augmentation therapy reduces frequency of lung infections in

Lind, P, et al. (2004). Risk of myocardial infarction and stroke in smokers is related to

Liu, HM, Takagaki, K & Schmid, K. (1988). In vitro nerve-growth-promoting activity of

Liu, Z, et al. (2000). The serpin alpha1-proteinase inhibitor is a critical substrate for

Liu, C, et al. (2005). Proapoptotic, antimigratory, antiproliferative, and antiangiogenic effects

Lu X, LY, et al. (2009). Decay-Accelerating Factor Attenuates C-Reactive Protein-Potentiated

Luo, LY, et al. (2006). Characterization of human kallikreins 6 and 10 in ascites fluid from

human plasma alpha 1-acid glycoprotein. *J Neurosci Res* 20, 64-72.

chemotactic protein-1 from lung epithelial cells: potential role in emphysema. *Am J*

sera from patients with probable late onset Alzheimer's disease*. Dement Geriatr*

antitrypsin deficiency: a new hypothesis with supporting data. *Chest* 118, 1480-

plasma levels of inflammation-sensitive proteins. *Arterioscler Thromb Vasc Biol* 24,

of commercial C-reactive protein on various human endothelial cell types in vitro: implications of contaminating presence of sodium azide in commercial preparation.

Tissue Injury After Mesenteric Ischemia/Reperfusion. *J Surg Res* [Epub ahead of

tumor necrosis factor-induced lethality. *J Exp Med* 180, 1571-1575.

mouse calvarial bones in vitro. *Arthritis Rheum* 35, 587-591.

Libby ,P. (2002). Inflammation in atherosclerosis. *Nature* 420, 868-874.

*Physiol Lung Cell Mol Physiol* 297, L388-400.

gelatinase B/MMP-9 in vivo. *Cell* 102, 647-655.

ovarian cancer patients. *Tumour Biol* 27, 227-234.

Boca Raton, FL.

412-420.

1485.

577-582.

print].

*Immunochemistry* 11, 703-709.

*Immunol* 157, 5126-5129.

*Cogn Disord* 8, 337-342.

*Circ Res* 97*,* 135-143.

*Proteins Molecular Biology, Biochemistry and Clinical Applications* pp: 3-19. CRC Press,

antichymotrypsin and alpha 1-proteinase inhibitor on NK cytotoxicity: only alpha

immunoglobulins and alpha 1-antitrypsin respectively prealbumin.

induces vascular cell adhesion molecule-1 expression through CD32 in human umbilical vein endothelial cells and aortic endothelial cells. *J Mol Cell Cardiol* 40,

Acute Phase Proteins: Structure and Function Relationship 57

Rinaldi, M, et al. (2008). Differential effects of alpha1-acid glycoprotein on bovine neutrophil

Rodriguez, W, et al. (2005). Reversal of ongoing proteinuria in autoimmune mice by

Rodriguez, W, et al. (2007). C-reactive protein-mediated suppression of nephrotoxic

Rossbacher, J, Wagner, L, & Pasternack, MS. (1999). Inhibitory effect of haptoglobin on

Roum, JH, et al. (1993). Systemic deficiency of glutathione in cystic fibrosis. *J Appl Physiol* 75,

Rydén, I, et al. (1997). Glycosylation of alpha1-acid glycoprotein in inflammatory disease:

Sadrzadeh, SM, & Bozorgmeh, J. (2004). Haptoglobin phenotypes in health and disorders.

Salvatore A, et al. (2009). Haptoglobin binds apolipoprotein E and influences cholesterol

Saldova, R, et al. (2008). Glycosylation changes on serum glycoproteins in ovarian cancer

Samberg, NL, et al. (1988). Preferential expression of neo-CRP epitopes on the surface of

Sarrats, A, et al. (2010). Glycosylation of liver acute-phase proteins in pancreatic cancer and

Scott, LJ, et al. (1998). Comparison of IgA-alpha1-antitrypsin levels in rheumatoid arthritis

Sei, K, et al. (2002). Collection of alpha1-acid glycoprotein molecular species by capillary

Shah, C, Hari-Dass, R & Raynes, JG. (2006). Serum amyloid A is an innate immune opsonin

Shapira, I, et al. (2011). Novel peptides as potential treatment of systemic lupus

Schapira, M, et al. (1982). Purified human plasma kallikrein aggregates human blood

She QB, et al. (2000). alpha(1)-antitrypsin can increase insulin-induced mitogenesis in

Schwedler, SB, et al. (2006). C-reactive protein: a family of proteins to regulate

Singh, VK & Fudenberg, HH. (1986). Lymphocyte stimulation in vitro by orosomucoid

Snyder, S & Coodley, EL. (1976). Inhibition of platelet aggregation by alpha1-acid

various fibroblast and epithelial cell lines. *FEBS Lett* 473, 33-36.

cardiovascular function. *Am J Kidney Dis* 47, 212-222.

and seronegative oligoarthritis: complex formation is not associated with

electrophoresis and the analysis of their molecular masses and carbohydrate chains. Basic studies on the analysis of glycoprotein glycoforms. *J Chromatogr A* 958, 273-

esterification in the cerebrospinal fluid.*J Neurochem*. 110, 255-263.

may contribute to disease pathogenesis. *Dis Markers* 25, 219-232.

human peripheral blood lymphocytes. *Cell Immunol* 116, 86-98.

chronic pancreatitis. *Proteomics Clin Appl* 4, 432-448.

inflammation per se. *Br J Rheumatol* 37, 398-404.

for Gram-negative bacteria. *Blood* 108, 1751-1757.

erythematosus. *Lupus [Epub ahead of print].*

neutrophils. *J Clin Invest* 69, 1199-1202.

glycoprotein. *Immunol Lett* 14, 9-13.

glycoprotein. *Arch Intern Med* 136, 778-781.

treatment with C-reactive protein. *Arthritis Rheum* 52, 642-650.

affinity immunoelectrophoresis. *Glycoconj J* 14, 481-488.

210.

178, 530-538.

399-404.

281.

2419-2424.

*Am J Clin Pathol* 121 *Suppl*.

respiratory burst activity and IL-8 production. *Vet Immunol Immunopathol* 126, 199-

nephritis: role of macrophages, complement, and Fcgamma receptors. *J Immunol*

granulocyte chemotaxis, phagocytosis and bactericidal activity. *Scand J Immunol* 50,

analysis by high-pH anion-exchange chromatography and concanavalin A crossed

Nielsen, MJ, & Moestrup, SK. (2009). Receptor targeting of hemoglobin mediated by the

Oh, SK, Pavlotsky, N, & Tauber, AI. (1990). Specific binding of haptoglobin to human neutrophils and its functional consequences. *J Leukoc Biol* 47, 142-148. Oliveira, EB, Gotschlich, C & Liu, TY. (1979). Primary structure of human C-reactive protein.

Okemefuna, AI, et al. (2010). C-reactive protein exists in an NaCl concentration-dependent pentamer-decamer equilibrium in physiological buffer. *J Biol Chem* 285, 1041-1052. Ossanna, PJ, et al. (1986). Oxidative regulation of neutrophil elastase-alpha-1-proteinase

Pagano, M, Nicola, MA, & Engler, R. (1982). Inhibition of cathepsin L and B by haptoglobin,

Pasceri, V, et al. (2001). Modulation of C-reactive protein-mediated monocyte

Pradhan, AD, et al. (2001). C-reactive protein, interleukin 6, and risk of developing type 2

Pasceri, V, Willerson, JT, & Yeh, ET. (2000). Direct proinflammatory effect of C-reactive

Pepys, MB, et al. (1994). Binding of pentraxins to different nuclear structures: C-reactive

component binds to chromatin and nucleoli. *Clin Exp Immunol* 97, 152-157. Petrache, I, et al. (2006). alpha-1 antitrypsin inhibits caspase-3 activity, preventing lung

Pichler, L, Muchitsch,EM & Schwarz, HP. (1999). [Preclinical investigation of alpha 1-acid

Poland, DC, et al. (2001). Increased alpha3-fucosylation of alpha1-acid glycoprotein in Type I diabetic patients is related to vascular function. *Glycoconj J* 18, 261-268. Potempa, LA, et al. (1983). Antigenic,electrophoretic and binding alterations of human C-

Potempa, LA, et al. (1996). Stimulation of megakaryocytopoiesis in mice by human modified

Potempa, LA, et al. (1987). Expression, detection and assay of a neoantigen (Neo-CRP) associated with a free, human C-reactive protein subunit. *Mol Immunol* 24, 531-541. Potthoff, AV, et al. (2007). HIV infection in a patient with alpha-1 antitrypsin deficiency: a

Rapala-Kozik, M, et al. (1999). Comparative cleavage sites within the reactive-site loop of

Retamales, I, et al. (2001). Amplification of inflammation in emphysema and its association with latent adenoviral infection. *Am J Respir Crit Care Med* 164, 469-473. Ridker, PM, et al. (1997). Alu-repeat polymorphism in the gene coding for tissue-type

native and oxidized alpha1-proteinase inhibitor by selected bacterial proteinases.

plasminogen activator (t-PA) and risks of myocardial infarction among middle-

protein on human endothelial cells. *Circulation* 102, 2165-2168.

glycoprotein (orosomucoid)]. *Wien Klin Wochenschr* 111, 192-198.

endothelial cell apoptosis. *Am J Pathol* 169, 1155-1166.

C-reactive protein (mCRP). *Exp Hematol* 24, 258-264.

aged men. *Arterioscler Thromb Vasc Biol* 17, 1687-1690.

detrimental combination? *AIDS* 21, 2115-2116.

*Biol Chem* 380, 1211-1216.

the haptoglobin-hemoglobin complex, and asialohaptoglobin. "In vitro" studies in

chemoattractant protein-1 induction in human endothelial cells by anti-

protein binds to small nuclear ribonucleoprotein particles, serum amyloid P

reactive protein modified selectively in the absence of calcium. *Mol Immunol* 20,

haptoglobins: roles beyond heme scavenging. *Blood* 114, 764-771.

inhibitor interactions. *J Clin Invest* 77, 1939-1951.

atherosclerosis drugs. *Circulation* 103, 2531-2534.

*J Biol Chem* 254, 489-502.

1165-1175.

the rat. *Can J Biochem* 60, 631-637.

diabetes mellitus. *JAMA* 286, 327-334.

Acute Phase Proteins: Structure and Function Relationship 59

Taylor, KE, Giddings, JC, & van den Berg, CW. (2005). C-reactive protein-induced in vitro

Thompson, D, Pepys, MB, & Wood, SP. (1999). The physiological structure of human Creactive protein and its complex with phosphocholine. *Structure* 7, 169-177. Thomas-Rudolph, D, et al. (2007). C-reactive protein enhances immunity to Streptococcus

Thurberg, BL, & Collins, T. (1998). The nuclear factor-kappa B/inhibitor of kappa B autoregulatory system and atherosclerosis. *Curr Opin Lipidol* 9, 387-396. Tilg H, et al. (1993) Antiinflammatory properties of hepatic acute phase proteins:

Tseng, CF, et al. (2004). Antioxidant role of human haptoglobin. *Proteomics* 4, 2221-2228. Tseng, IC, et al. (2008). Purification from human milk of matriptase complexes with secreted

Valko, M, et al. (2007). Free radicals and antioxidants in normal physiological functions and

Van Molle, W, et al. (1999). Activation of caspases in lethal experimental hepatitis and

van Pelt, J, et al. (1996). Carbohydrate-deficient transferrin values in neonatal and umbilical

Verma, S, et al. (2002). Endothelin antagonism and interleukin-6 inhibition attenuate the proatherogenic effects of C-reactive protein. *Circulation* 105, 1890-1896. Volanakis, JE, & Wirtz, KW. (1979). Interaction of C-reactive protein with artificial

Walcheck, B, et al. (1996). Neutrophil-neutrophil interactions under hydrodynamic shear

Wang, M, et al. (2009). Novel fucosylated biomarkers for the early detection of hepatocellular carcinoma. *Cancer Epidemiol Biomarkers Prev* 18, 1914-1921. Weiser ,JN, et al. (1998). Phosphorylcholine on the lipopolysaccharide of Haemophilus

Whisler, RL, Newhouse, YG, & Mortensen, RF. (1986). C-reactive protein reduces the

Wiman, B, et al. (1988). Plasminogen activator inhibitor 1 (PAI) is bound to vitronectin in

Williams JP, et al. (1997). Alpha1-Acid glycoprotein reduces local and remote injuries after

Woollard, KJ, Phillips, DC, & Griffiths, HR. (2002). Direct modulatory effect of C-reactive

serum killing mediated by C-reactive protein. *J Exp Med* 187, 631-640.

stress involve L-selectin and PSGL-1. A mechanism that amplifies initial leukocyte

influenzae contributes to persistence in the respiratory tract and sensitivity to

promotion of human B-cell colony formation by autoreactive T4 cells and T-cell proliferation during the autologous mixed-lymphocyte reaction. *Cell Immunol* 102,

protein on primary human monocyte adhesion to human endothelial cells. *Clin Exp*

prevention by acute phase proteins. *J Immunol* 163, 5235-5241.

accumulation of P-selectin in vitro. *J Clin Invest* 98, 1081-1087.

Weiss, SJ. (1989). Tissue destruction by neutrophils *N Engl J Med* 320, 365-376.

intestinal ischemia in the rat. *Am J Physiol* 273, G1031-10335.

*Arterioscler Thromb Vasc Biol* 25, 1225-1230.

human disease. *Int J Biochem Cell Biol.* 39, 44-84.

phosphatidylcholine bilayers. *Nature* 281, 155-157.

cord blood. *J Inherit Metab Dis* 19, 253-266.

7283-7291.

287-298.

plasma. *FEBS Lett* 242, 125-128.

*Immunol Today* 130, 256-262.

*Physiol* 295, C423-C431.

endothelial cell activation is an artefact caused by azide and lipopolysaccharide.

pneumoniae by targeting uptake to Fc gamma R on dendritic cells. *J Immunol* 178,

preferential induction of interleukin 1 (IL-1) receptor antagonist over IL-1 beta synthesis by human peripheral blood mononuclear cells. *J Exp Med* 178, 1629-1636.

serpins: mechanism for inhibition of matriptase other than HAI-1. *Am J Physiol Cell*

Stefansson, S, & Lawrence, DA. (1996). The serpin PAI-1 inhibits cell migration by blocking

Steinmetz, A, et al. (1989). Influence of serum amyloid A on cholesterol esterification in

Stibler, H, Holzbach, U & Kristiansson, B. (1998). Isoforms and levels of transferrin,

Stoller, JK, et al. (2007). Primary care diagnosis of alpha-1 antitrypsin deficiency: issues and

Stonik, JA, et al. (2004). Serum amyloid A promotes ABCA1-dependent and ABCA1 independent lipid efflux from cells. *Biochem Biophys Res Commun* 32, 936-941. Su, SJ, et al. (1999). Alpha 1-acid glycoprotein-induced tumor necrosis factor-alpha secretion

Su, SJ & Yeh, TM. (1996). Effects of alpha 1-acid glycoprotein on tissue factor expression and

Sun, YX, Wright, HT, & Janciauskiene, S. (2002). Alpha1-antichymotrypsin/Alzheimer's

Subramaniyam, D., et al. (2010). Cholesterol rich lipid raft microdomains are gateway for acute phase protein, SERPINA1*. Int J Biochem Cell Biol* 42, 1562-1570. Subramaniyam, D, et al. (2008). TNF-alpha-induced self expression in human lung

Subramaniyam, D., et al. (2010). Effects of alpha 1-antitrypsin on endotoxin-induced lung

Sullivan, CP, et al. (2010). Secretory phospholipase A2, group IIA is a novel serum amyloid

Szalai, AJ, Briles, DE & Volanakis, JE. (1995). Human C-reactive protein is protective against

Szalai, AJ, et al. (2002). Experimental allergic encephalomyelitis is inhibited in transgenic mice expressing human C-reactive protein. *J Immunol* 168, 5792-5797. Szalai, AJ, et al. (2000). Human C-reactive protein is protective against fatal Salmonella

Sörensson, J, et al. (1999). Human endothelial cells produce orosomucoid, an important

Tam, SP, Kisilevsky, R, & Ancsin, JB. (2008). Acute-phase-HDL remodeling by heparan

Taggart, C, et al. (2000). Oxidation of either methionine 351 or methionine 358 in alpha 1-

component of the capillary barrier. *Am J Physiol* 276, H530-534.

protein tyrosine kinase activation. *Immunopharmacology.* 41, 21-29.

antithrombin, alpha(1)-antitrypsin and thyroxine-binding globulin in 48 patients with carbohydrate-deficient glycoprotein syndrome type I. *Scand J Clin Lab Invest*

of human monocytes is enhanced by serum binding proteins and depends on

tumor necrosis factor secretion in human monocytes. *Immunopharmacology* 34, 139-

peptide Abeta(1-42) complex perturbs lipid metabolism and activates transcription factors PPARgamma and NFkappaB in human neuroblastoma (Kelly) cells. *J*

endothelial cells is inhibited by native and oxidized alpha1-antitrypsin*. Int J*

A target gene: activation of smooth muscle cell expression by an interleukin-1

fatal Streptococcus pneumoniae infection in transgenic mice. *J Immunol* 155, 2557-

enterica serovar typhimurium infection in transgenic mice. *Infect Immun* 68, 5652-

sulfate generates a novel lipoprotein with exceptional cholesterol efflux activity

antitrypsin causes loss of anti-neutrophil elastase activity*. J Biol Chem* 275, 27258-

integrin alpha V beta 3 binding to vitronectin. *Nature* 383, 441-443.

human plasma. *Biochim Biophys Acta* 1006, 173-178.

opportunities. *Cleve Clin J Med* 74, 869-874.

*Neurosci Respir Med* 67, 511-522.

*Biochem Cell Biol* 40, 258-271.

inflammation in vivo*. Inflamm Res* 59, 571-578.

from macrophages. *PLoS One* 3, e3867.

receptor-independent mechanism. *J Biol Chem* 285, 565-575.

58, 55-61.

145.

2563.

5656.

27265.

**3**

*Poland*

**Regulatory Mechanisms Controlling**

Inflammation is usually defined as a complex response of the animal organism to tissue injury or to invasion of foreign pathogens. Although inflammation often starts as a local reaction it can easily develop into a generalized systemic response involving the whole organism. Participation of inflammatory reactions in the pathogenesis of many diseases is today a generally accepted medical paradigm. These ideas have been thoroughly discussed in numerous excellent monographs and specialized recent publications (Nathan, 2002; Dinarello, 2008, 2010; Medzhitov, 2010) whereas our presentation is just a brief account focused on regulatory mechanisms involved in the initiation, development and termination of the "generalized inflammatory response" (**GIR**) and its relations to the induced synthesis

The results obtained by techniques of modern genomics indicate that these three phases of response to injury are tightly controlled and are characterized by distinct profiles of hundreds of activated genes. The fine tuning of gene expression during initiation, development and resolution of inflammation occurs at many levels starting from transcription and followed by changes in mRNA decay, receptors cross-talk, intracellular signaling cascades and posttranslational protein modifications. Disturbances in the regulatory network lead to either acute pathological states such as septic shock, or to

Discovery of C-reactive protein (**CRP**) in the blood of patients with febrile diseases some 80 years ago (Tillett & Francis, 1930) was seminal for the development of the idea of "acute phase reaction" (**APR**) and the concept of "acute phase proteins" (**APPs**) reflected as profound rearrangement of plasma protein profile accompanying systemic inflammatory reaction (Kushner, 1982). The so called "positive AP-proteins" increase their plasma concentration in a broad range – from barely 25 per cent above the control up to several hundred fold - at the expense of "negative APPs" which go down in comparison to control. Other metabolic changes during this response include fever, leukocytosis, negative nitrogen balance, altered levels of some ions and hormones, activation of clotting and complement pathways, as well as certain less defined phenomena (for references see (Gordon and Koj, 1985)). The principal features of APR resemble the unspecific innate immunological reaction

**1. Introduction**

of certain plasma and tissue proteins.

various chronic autoimmune and auto-inflammatory diseases.

**Inflammation and Synthesis**

*Faculty of Biochemistry, Biophysics and Biotechnology*

**of Acute Phase Proteins**

Jolanta Jura and Aleksander Koj

*Jagiellonian University*, *Krakow*,

## **Regulatory Mechanisms Controlling Inflammation and Synthesis of Acute Phase Proteins**

Jolanta Jura and Aleksander Koj

*Faculty of Biochemistry, Biophysics and Biotechnology Jagiellonian University*, *Krakow*, *Poland*

#### **1. Introduction**

60 Acute Phase Proteins – Regulation and Functions of Acute Phase Proteins

Wong, PS, & Travis, J. (1980). Isolation and properties of oxidized alpha-1-proteinase

Xia, D & Samols, D. (1997). Transgenic mice expressing rabbit C-reactive protein are

Xie, L, et al. (2005). C-reactive protein augments interleukin-8 secretion in human peripheral

Yang, F, et al. (1995). Cell type-specific and inflammatory-induced expression of

Yang, Z & Hancock, WS. (2005). Monitoring glycosylation pattern changes of glycoproteins using multi-lectin affinity chromatography. *J Chromatogr A* 1070, 57-64. Zeller, JM, & Sullivan, BL. (1992). C-reactive protein selectively enhances the intracellular

Zhang, WM, et al. (2000). Characterization and determination of the complex between

Zhong, W, et al. (1998) Effect of human C-reactive protein on chemokine and chemotactic factor-induced neutrophil chemotaxis and signaling. *J Immunol* 161, 2533-2540. Zipfel, PF & Skerka, C. (2009). Complement regulators and inhibitory proteins. *Nat Rev*

Zouki, C, et al. (1997). Prevention of In vitro neutrophil adhesion to endothelial cells

Zouki, C, et al. (2001). Loss of pentameric symmetry of C-reactive protein is associated with promotion of neutrophil-endothelial cell adhesion. *J Immunol* 167, 5355-5361.

generation of reactive oxygen products by IgG-stimulated monocytes and

prostate-specific antigen and alpha 1-protease inhibitor in benign and malignant

through shedding of L-selectin by C-reactive protein and peptides derived from C-

resistant to endotoxemia.  *J Cardiovasc Pharmacol* 46, 690-696.

prostatic diseases. *Scand J Clin Lab Invest Suppl* 233, 51-58.

haptoglobin gene in lung. *Lab Invest. 73*, 433-440.

neutrophils. *J Leukoc Biol* 52, 449-455.

reactive protein. *J Clin Invest* 100, 522-529.

*Immunol* 9, 729-740.

1449-1454.

inhibitor from human rheumatoid synovial fluid. *Biochem Biophys Res Commun* 96,

Inflammation is usually defined as a complex response of the animal organism to tissue injury or to invasion of foreign pathogens. Although inflammation often starts as a local reaction it can easily develop into a generalized systemic response involving the whole organism. Participation of inflammatory reactions in the pathogenesis of many diseases is today a generally accepted medical paradigm. These ideas have been thoroughly discussed in numerous excellent monographs and specialized recent publications (Nathan, 2002; Dinarello, 2008, 2010; Medzhitov, 2010) whereas our presentation is just a brief account focused on regulatory mechanisms involved in the initiation, development and termination of the "generalized inflammatory response" (**GIR**) and its relations to the induced synthesis of certain plasma and tissue proteins.

The results obtained by techniques of modern genomics indicate that these three phases of response to injury are tightly controlled and are characterized by distinct profiles of hundreds of activated genes. The fine tuning of gene expression during initiation, development and resolution of inflammation occurs at many levels starting from transcription and followed by changes in mRNA decay, receptors cross-talk, intracellular signaling cascades and posttranslational protein modifications. Disturbances in the regulatory network lead to either acute pathological states such as septic shock, or to various chronic autoimmune and auto-inflammatory diseases.

Discovery of C-reactive protein (**CRP**) in the blood of patients with febrile diseases some 80 years ago (Tillett & Francis, 1930) was seminal for the development of the idea of "acute phase reaction" (**APR**) and the concept of "acute phase proteins" (**APPs**) reflected as profound rearrangement of plasma protein profile accompanying systemic inflammatory reaction (Kushner, 1982). The so called "positive AP-proteins" increase their plasma concentration in a broad range – from barely 25 per cent above the control up to several hundred fold - at the expense of "negative APPs" which go down in comparison to control. Other metabolic changes during this response include fever, leukocytosis, negative nitrogen balance, altered levels of some ions and hormones, activation of clotting and complement pathways, as well as certain less defined phenomena (for references see (Gordon and Koj, 1985)). The principal features of APR resemble the unspecific innate immunological reaction

Regulatory Mechanisms Controlling Inflammation and Synthesis of Acute Phase Proteins 63

RNA from viruses and endogenous sources. Finally, TLR9 recognizes DNA-containing unmethylated CpG motifs. All the discussed TLRs differ not only in structure and ligand affinity but also in the intracellular signaling cascades. Their ability to form dimers certainly expands the array of recognized PAMPs. Signals generated by the formation of a PAMP-TLR complex activate at least one out of five specific adaptor cytoplasmic proteins (MyD88, MAL, TRIF, TRAF or SARM) (O'Neill & Bowie, 2007). These adaptors participate in transmission of a message from the plasma membrane through a multi-step cascade to a responsive

Fig. 1. Scheme of initiation and development of inflammatory response. For abbreviations

used and for further detail see the text.

transcription factor – NF-κB being the principal target (Fig.1).

and some authors assume that in fact we are dealing with synonyms corresponding to the two sides of the same biological phenomenon (Koj, 2008). From historic perspective however, the APR corresponds to the metabolic response of liver, manifested predominantly as induced synthesis of acute phase proteins. For several years it was silently assumed that the term APP is limited to plasma proteins synthesized by and secreted from liver parenchymal cells. However, many resident proteins are also affected by inflammatory response and they are called "acute phase regulated intracellular proteins" i.e. APRIPs (Ruminy et al., 2001), or even more broadly – cytokine-responsive cellular proteins (Koj, 2008).

#### **2. Initiation of generalized inflammatory response**

Inflammation can be induced by two types of noxious stimuli: invasion of foreign pathogens of biological origin, or sterile tissue damage. In reality, however, the most common is mixed type of GIR. The pathogens include bacteria, fungi, yeasts, viruses or parasites as well as their products which after penetrating of the organism are present either in the extracellular space, or already in the cell compartments. In the first case they can be recognized by a special class of cell membrane receptors abounding on the surface of sentinel cells exposed to the environment. Receptors of this type were first discovered in *Drosophila* fruit fly and named Toll receptors, being important for embryonic development and protection against invading fungi. Their mammalian counterparts named Toll-like receptors **(TLRs)** have been extensively studied during last 15 years (Medzhitov et al., 1997). TLRs are able to discriminate between self and non-self by recognizing pathogen-associated molecular patterns **(PAMPs)** (Medzhitov, 2010). Typical examples of this class of PAMPs are lipopolysaccharides of the bacterial wall, or mannans from the yeasts cells (Takeuchi & Akira, 2010). Today more than ten mammalian TLRs are known and depending on their cellular localization and type of the ligand they are divided into two subgroups. TLRs from the first group are localized at the cell membrane (TLRs 1, 2, 4, 5, 6, 10), whereas others (TLRs 3, 7, 8, 9) are expressed intracellularly anchoring vesicles of the endoplasmic reticulum, endosomes and lysosomes (Kawai & Akira, 2010; Ospelt & Gay, 2010). Toll-like receptors are important in the initiation of molecular alarm during pathogen invasion of the host and also in response to other danger signals expressed in a variety of immune as well as non-immune cells. Effector or "sentinel" cells, such as neutrophils, dendritic cells and macrophages express almost all types of TLRs, in distinction to other specialized cells. The first group of TLRs recognizes mainly microbial membrane components (lipids, lipoproteins and proteins) whereas the other group responds to microbial nucleic acids. For example TLR1 acts mainly as a heterodimer together with TLR2 and recognizes lipopeptides originating from *Mycobacteria*. TLR2 responds also to material derived from bacteria, fungi, parasites and viruses (lipopeptides, peptidoglycan, lipoarabinomannan, zymosan or hemagglutinins), whereas the heterodimer TLR2/TLR6 binds lipopeptides. TLR4 was the first TLR identified in human cells and we know that it responds to bacterial lipopolysaccharides (LPS) from the membrane of Gram-negative bacteria (Akira et al., 2006). Another receptor from this group, TLR5, recognizes bacterial protein, flagellin, essential for movement of bacteria (Akira et al., 2006). The recently discovered human TLR10 was found to heterodimerize with TLR1 (Hasan et al., 2005) but so far its ligand was not determined. As already mentioned, TLRs from intracellular compartments may recognize nucleic acids: TLR3 binds double stranded RNA of viral origin, but also similar endogenous ligands from necrotic cells (Brentano et al., 2005). On the other hand, TLR7 and TLR8 respond to single-stranded

and some authors assume that in fact we are dealing with synonyms corresponding to the two sides of the same biological phenomenon (Koj, 2008). From historic perspective however, the APR corresponds to the metabolic response of liver, manifested predominantly as induced synthesis of acute phase proteins. For several years it was silently assumed that the term APP is limited to plasma proteins synthesized by and secreted from liver parenchymal cells. However, many resident proteins are also affected by inflammatory response and they are called "acute phase regulated intracellular proteins" i.e. APRIPs (Ruminy et al., 2001), or even more broadly – cytokine-responsive cellular proteins (Koj,

Inflammation can be induced by two types of noxious stimuli: invasion of foreign pathogens of biological origin, or sterile tissue damage. In reality, however, the most common is mixed type of GIR. The pathogens include bacteria, fungi, yeasts, viruses or parasites as well as their products which after penetrating of the organism are present either in the extracellular space, or already in the cell compartments. In the first case they can be recognized by a special class of cell membrane receptors abounding on the surface of sentinel cells exposed to the environment. Receptors of this type were first discovered in *Drosophila* fruit fly and named Toll receptors, being important for embryonic development and protection against invading fungi. Their mammalian counterparts named Toll-like receptors **(TLRs)** have been extensively studied during last 15 years (Medzhitov et al., 1997). TLRs are able to discriminate between self and non-self by recognizing pathogen-associated molecular patterns **(PAMPs)** (Medzhitov, 2010). Typical examples of this class of PAMPs are lipopolysaccharides of the bacterial wall, or mannans from the yeasts cells (Takeuchi & Akira, 2010). Today more than ten mammalian TLRs are known and depending on their cellular localization and type of the ligand they are divided into two subgroups. TLRs from the first group are localized at the cell membrane (TLRs 1, 2, 4, 5, 6, 10), whereas others (TLRs 3, 7, 8, 9) are expressed intracellularly anchoring vesicles of the endoplasmic reticulum, endosomes and lysosomes (Kawai & Akira, 2010; Ospelt & Gay, 2010). Toll-like receptors are important in the initiation of molecular alarm during pathogen invasion of the host and also in response to other danger signals expressed in a variety of immune as well as non-immune cells. Effector or "sentinel" cells, such as neutrophils, dendritic cells and macrophages express almost all types of TLRs, in distinction to other specialized cells. The first group of TLRs recognizes mainly microbial membrane components (lipids, lipoproteins and proteins) whereas the other group responds to microbial nucleic acids. For example TLR1 acts mainly as a heterodimer together with TLR2 and recognizes lipopeptides originating from *Mycobacteria*. TLR2 responds also to material derived from bacteria, fungi, parasites and viruses (lipopeptides, peptidoglycan, lipoarabinomannan, zymosan or hemagglutinins), whereas the heterodimer TLR2/TLR6 binds lipopeptides. TLR4 was the first TLR identified in human cells and we know that it responds to bacterial lipopolysaccharides (LPS) from the membrane of Gram-negative bacteria (Akira et al., 2006). Another receptor from this group, TLR5, recognizes bacterial protein, flagellin, essential for movement of bacteria (Akira et al., 2006). The recently discovered human TLR10 was found to heterodimerize with TLR1 (Hasan et al., 2005) but so far its ligand was not determined. As already mentioned, TLRs from intracellular compartments may recognize nucleic acids: TLR3 binds double stranded RNA of viral origin, but also similar endogenous ligands from necrotic cells (Brentano et al., 2005). On the other hand, TLR7 and TLR8 respond to single-stranded

**2. Initiation of generalized inflammatory response**

2008).

RNA from viruses and endogenous sources. Finally, TLR9 recognizes DNA-containing unmethylated CpG motifs. All the discussed TLRs differ not only in structure and ligand affinity but also in the intracellular signaling cascades. Their ability to form dimers certainly expands the array of recognized PAMPs. Signals generated by the formation of a PAMP-TLR complex activate at least one out of five specific adaptor cytoplasmic proteins (MyD88, MAL, TRIF, TRAF or SARM) (O'Neill & Bowie, 2007). These adaptors participate in transmission of a message from the plasma membrane through a multi-step cascade to a responsive transcription factor – NF-κB being the principal target (Fig.1).

Fig. 1. Scheme of initiation and development of inflammatory response. For abbreviations used and for further detail see the text.

Regulatory Mechanisms Controlling Inflammation and Synthesis of Acute Phase Proteins 65

able to discriminate between self and non-self of certain molecular structures that may appear either outside of the cell (e.g. bacterial LPS is bound to Toll-like receptors located on the cell membrane), or are found intracellularly (e.g. foreign DNA or RNA in the cytosol) where they are recognized by NOD-like receptors (NLRs). However, PRRs can be stimulated not only by binding Pathogen Associated Molecular Patterns (**PAMPs**) but also by host own stress and danger signals (**SAMPs and DAMPs**) derived from damaged cells. These signals are recognized by cytosolic, intracellular receptors, among which the most important are NLRs. Moreover, some NLRs are used for the construction of specialized subcellular structures – inflammasomes – serving as the activation platform for the key pro-

Undoubtedly, IL-1β is the key mediator in the host innate response to infection and the driving force in the development of inflammation. However, since IL-1β is synthesized as a leaderless pro-cytokine it must be processed for secretion and exhibition of biological activity (similar properties are shown also by IL-18 and IL-33) (Dinarello, 1996, 2008, 2010). The proteolytic activation of pro-IL-1β is accomplished by an enzyme initially named ICE (Interleukin-1-converting enzyme) but at present known as caspase-1 belonging to the family of cysteine-aspartyl proteases participating in apoptosis. Caspase-1 is synthesized as zymogen which undergoes autocatalytic processing. Only relatively recently it has been established that efficient activation of pro-caspase-1 and pro-IL-1β takes place in specialized cytoplasmic multiprotein platforms called **inflammasomes** (Dinarello, 2008; Martinon et al.,

As discussed by Martinon et al. (2009), the details of inflammasome structure are only partly revealed but several types of molecules can be distinguished. The best known NLRP3 inflammasome (alternative names – NALP3 or cryopyrin) includes NLRP3 protein which interacts with the **adaptor protein ASC** (apoptosis-associated speck-like protein with a caspase recruitment domain). The resulting complex binds and activates pro-caspase-1 to caspase-1 which in turn is ready to activate pro-IL-1β (Fig.2). It appears that activation of caspase-1 requires zinc and is dependent on pannexin-1, a protein localized upstream in this

Assembly and activation of inflammasome can be induced by several factors discussed above, but recent review presented by Martinon et al., (2009) enlarges this list to include various signals of danger and stress (DAMPs or SAMPs). Extracellular ATP represents such a stress signal released locally and recognized by a specific purinergic receptor P2X7 (Ferrari

Charles Dinarello was probably first to report that in order to obtain active and mature IL-1β molecules two independent cellular signals are required for effective transcription and translation (Dinarello, 1996). When studying activation of NLRP3 inflammasome Bauernfeind et al., (2009) observed that this process also needs two signals: one provided by the NF-κB cascade is necessary but not sufficient, whereas the second signal is generated by extracellular ATP, or crystal-induced cell damage. Other inducers of inflammasome assembly include such widely different factors as reactive oxygen intermediates, products of lysosomal damage and leakage of potassium from the cell (Martinon et al., 2009; Rock et al., 2010). We know today that the inflammasome may act as a sensor of the oxidative stress by co-operation with thioredoxin (Jin & Flavell, 2010; Zhou et al., 2010). The broad spectrum of

inflammatory cytokine – IL-1β.

**3. The role of inflammasomes**

2009; Schroder & Tschopp, 2010).

signaling cascade (Brough et al., 2009).

et al., 2006; Lister et al., 2007).

However, some pathogen-derived molecules, such as foreign DNA or RNA, may reach intracellular compartments of infected cells where they are recognized by and bound to NOD-like receptors (**NLRs**) (Akira et al., 2006; Ye & Ting, 2008). The NLRs belong to a large family of cytosolic pattern recognition receptors (34 members in mice, 23 in human) (Jin & Flawell, 2010) that are able to recognize various pathogen associated molecular patterns or danger-associated molecular patterns and thereby initiate the innate immune response against invading pathogens and cellular signals of damage or stress.

These NLRs, multi-domain receptor proteins, are characterized by leucine-reach repeats (LRRs) at the C-terminal region, a central nucleotide binding and oligomerization domain (referred to as NOD domain), toward N-terminal effector domain. From evolutionary point of view it is very interesting that animal NLRs show certain similarities to the products of plant disease-resistant genes (Martinon et al., 2009). Some NLRs act just as receptors while other constitute components of specialized cytosolic structures – **inflammasomes** (Schroder & Tschopp, 2010; Martinon et al., 2009). Certain NLRs interact with the ubiquitin ligaseassociated protein SGT1 and HSP90, and this leads to the inhibition of inflammasome activity (Mayor et al. 2007).

In comparison to pathogen induced response the signaling elicited by pathogen-free tissue injury is even more complex and includes various **DAMPs** (damage-associated molecular patterns) such as crystals of uric acid or cholesterol (responsible for symptoms of gout or atherosclerosis) (Rock et al., 2010), fibers of asbestos and grains of silica (accumulated in lungs in asbestosis and silicosis) (Martinon et al., 2009; Rock et al., 2010), chemical irritants such as turpentine oil (Sheikh et al., 2007), deposits of denatured or modified proteins (such as inactivated alpha-1-proteinase inhibitor) (Koj & Guzdek, 1995), or fibrillar amyloid beta peptide found in the brain in Alzheimer disease (Halle et al., 2008). But commonly occurring pathogen-free tissue damage leading to inflammation can be also elicited by extreme temperatures, by hypo- or hyperosmolarity (Shapiro & Dinarello, 1997; Bode et al., 1999), by hypoxia-reoxygenation process (Wenger et al., 1995), UV radiation (Feldmeyer et al., (2007) and accumulation of reactive oxygen species (Bogdan et al., 2000). These signals may - by various means - stimulate cells either through specialized appropriate Toll-like receptors (Takeuchi & Akira, 2010) or certain scavenger receptors, or using other sensitive intracellular surveillance molecules, such as plasma membrane transporters (Schroder and Tchopp, 2010) or activation of MAP kinases (Bode et al., 1999; Chang & Karin, 2001). The use of alternative signaling routes occurs in case of hypoxia which is known to mobilize a specific transcription factor HIF-1 and proline hydroxylases (Zagorska & Dulak 2004; Oliver et al., 2009).

Usually the multi-step signaling cascade activated by pathogen-free tissue injury (or by stress of environmental origin) merges at some stage with the cascade initiated by foreign pathogens resulting in activation of transcription factors, among which the most important is NF-κB (Fig.1). This leads to a prompt transcription of sensitive genes resulting in accumulation of specific mRNAs coding mainly for IL-1β and other cytokines involved in the development of inflammation. It is understood now that translation of some of these mRNAs into active proteins may require an additional signal as it was earlier found in the case of IL-1β (Dinarello, 1996); if this signal is missing, initiation of the inflammatory response may be delayed or entirely aborted.

In order to briefly recapitulate current views on the initiation of inflammatory reaction one should emphasize the importance of signals indicating imminent danger to the cell/organism. Their detection is based on the ability of cells to recognize such signals by a small number of germline-encoded Pattern Recognition Receptors (PRRs)*.* These PRRs are able to discriminate between self and non-self of certain molecular structures that may appear either outside of the cell (e.g. bacterial LPS is bound to Toll-like receptors located on the cell membrane), or are found intracellularly (e.g. foreign DNA or RNA in the cytosol) where they are recognized by NOD-like receptors (NLRs). However, PRRs can be stimulated not only by binding Pathogen Associated Molecular Patterns (**PAMPs**) but also by host own stress and danger signals (**SAMPs and DAMPs**) derived from damaged cells. These signals are recognized by cytosolic, intracellular receptors, among which the most important are NLRs. Moreover, some NLRs are used for the construction of specialized subcellular structures – inflammasomes – serving as the activation platform for the key proinflammatory cytokine – IL-1β.

#### **3. The role of inflammasomes**

64 Acute Phase Proteins – Regulation and Functions of Acute Phase Proteins

However, some pathogen-derived molecules, such as foreign DNA or RNA, may reach intracellular compartments of infected cells where they are recognized by and bound to NOD-like receptors (**NLRs**) (Akira et al., 2006; Ye & Ting, 2008). The NLRs belong to a large family of cytosolic pattern recognition receptors (34 members in mice, 23 in human) (Jin & Flawell, 2010) that are able to recognize various pathogen associated molecular patterns or danger-associated molecular patterns and thereby initiate the innate immune

These NLRs, multi-domain receptor proteins, are characterized by leucine-reach repeats (LRRs) at the C-terminal region, a central nucleotide binding and oligomerization domain (referred to as NOD domain), toward N-terminal effector domain. From evolutionary point of view it is very interesting that animal NLRs show certain similarities to the products of plant disease-resistant genes (Martinon et al., 2009). Some NLRs act just as receptors while other constitute components of specialized cytosolic structures – **inflammasomes** (Schroder & Tschopp, 2010; Martinon et al., 2009). Certain NLRs interact with the ubiquitin ligaseassociated protein SGT1 and HSP90, and this leads to the inhibition of inflammasome

In comparison to pathogen induced response the signaling elicited by pathogen-free tissue injury is even more complex and includes various **DAMPs** (damage-associated molecular patterns) such as crystals of uric acid or cholesterol (responsible for symptoms of gout or atherosclerosis) (Rock et al., 2010), fibers of asbestos and grains of silica (accumulated in lungs in asbestosis and silicosis) (Martinon et al., 2009; Rock et al., 2010), chemical irritants such as turpentine oil (Sheikh et al., 2007), deposits of denatured or modified proteins (such as inactivated alpha-1-proteinase inhibitor) (Koj & Guzdek, 1995), or fibrillar amyloid beta peptide found in the brain in Alzheimer disease (Halle et al., 2008). But commonly occurring pathogen-free tissue damage leading to inflammation can be also elicited by extreme temperatures, by hypo- or hyperosmolarity (Shapiro & Dinarello, 1997; Bode et al., 1999), by hypoxia-reoxygenation process (Wenger et al., 1995), UV radiation (Feldmeyer et al., (2007) and accumulation of reactive oxygen species (Bogdan et al., 2000). These signals may - by various means - stimulate cells either through specialized appropriate Toll-like receptors (Takeuchi & Akira, 2010) or certain scavenger receptors, or using other sensitive intracellular surveillance molecules, such as plasma membrane transporters (Schroder and Tchopp, 2010) or activation of MAP kinases (Bode et al., 1999; Chang & Karin, 2001). The use of alternative signaling routes occurs in case of hypoxia which is known to mobilize a specific transcription factor HIF-1 and proline hydroxylases (Zagorska & Dulak 2004; Oliver

Usually the multi-step signaling cascade activated by pathogen-free tissue injury (or by stress of environmental origin) merges at some stage with the cascade initiated by foreign pathogens resulting in activation of transcription factors, among which the most important is NF-κB (Fig.1). This leads to a prompt transcription of sensitive genes resulting in accumulation of specific mRNAs coding mainly for IL-1β and other cytokines involved in the development of inflammation. It is understood now that translation of some of these mRNAs into active proteins may require an additional signal as it was earlier found in the case of IL-1β (Dinarello, 1996); if this signal is missing, initiation of the inflammatory

In order to briefly recapitulate current views on the initiation of inflammatory reaction one should emphasize the importance of signals indicating imminent danger to the cell/organism. Their detection is based on the ability of cells to recognize such signals by a small number of germline-encoded Pattern Recognition Receptors (PRRs)*.* These PRRs are

response against invading pathogens and cellular signals of damage or stress.

activity (Mayor et al. 2007).

et al., 2009).

response may be delayed or entirely aborted.

Undoubtedly, IL-1β is the key mediator in the host innate response to infection and the driving force in the development of inflammation. However, since IL-1β is synthesized as a leaderless pro-cytokine it must be processed for secretion and exhibition of biological activity (similar properties are shown also by IL-18 and IL-33) (Dinarello, 1996, 2008, 2010). The proteolytic activation of pro-IL-1β is accomplished by an enzyme initially named ICE (Interleukin-1-converting enzyme) but at present known as caspase-1 belonging to the family of cysteine-aspartyl proteases participating in apoptosis. Caspase-1 is synthesized as zymogen which undergoes autocatalytic processing. Only relatively recently it has been established that efficient activation of pro-caspase-1 and pro-IL-1β takes place in specialized cytoplasmic multiprotein platforms called **inflammasomes** (Dinarello, 2008; Martinon et al., 2009; Schroder & Tschopp, 2010).

As discussed by Martinon et al. (2009), the details of inflammasome structure are only partly revealed but several types of molecules can be distinguished. The best known NLRP3 inflammasome (alternative names – NALP3 or cryopyrin) includes NLRP3 protein which interacts with the **adaptor protein ASC** (apoptosis-associated speck-like protein with a caspase recruitment domain). The resulting complex binds and activates pro-caspase-1 to caspase-1 which in turn is ready to activate pro-IL-1β (Fig.2). It appears that activation of caspase-1 requires zinc and is dependent on pannexin-1, a protein localized upstream in this signaling cascade (Brough et al., 2009).

Assembly and activation of inflammasome can be induced by several factors discussed above, but recent review presented by Martinon et al., (2009) enlarges this list to include various signals of danger and stress (DAMPs or SAMPs). Extracellular ATP represents such a stress signal released locally and recognized by a specific purinergic receptor P2X7 (Ferrari et al., 2006; Lister et al., 2007).

Charles Dinarello was probably first to report that in order to obtain active and mature IL-1β molecules two independent cellular signals are required for effective transcription and translation (Dinarello, 1996). When studying activation of NLRP3 inflammasome Bauernfeind et al., (2009) observed that this process also needs two signals: one provided by the NF-κB cascade is necessary but not sufficient, whereas the second signal is generated by extracellular ATP, or crystal-induced cell damage. Other inducers of inflammasome assembly include such widely different factors as reactive oxygen intermediates, products of lysosomal damage and leakage of potassium from the cell (Martinon et al., 2009; Rock et al., 2010). We know today that the inflammasome may act as a sensor of the oxidative stress by co-operation with thioredoxin (Jin & Flavell, 2010; Zhou et al., 2010). The broad spectrum of

Regulatory Mechanisms Controlling Inflammation and Synthesis of Acute Phase Proteins 67

later by van den Berghe et al., (2000), may provide some useful information.

Fig. 3. Simplified scheme of a signaling pathway activated by bacterial endotoxin (LPS). LPS (1) interacts with LPS-binding protein (2) and with help of plasma membrane receptor CD14 (3) is transferred to Toll-like receptor TLR4 (5) which requires MD2 accessory protein (4). Activation of TLR4 leads to the recruitment of an adaptor protein MyD88 with subsequent involvement of several adaptor or enzymatic proteins (some of them not shown on the scheme): Tollip, IRAK (IL-1R-Associated Kinase) and TRAF (TNF-Receptor Associated Factor). IRAK4 is phosphorylated and activates IRAK1, which in turn interacts with TNF receptor-associated factor 6 (TRAF6) complex causing its oligomerization and activation. Proteins from TRAF6 complex activate IKK and MAP kinases (ERK1/3, JNK, p38). IKK is a complex of two protein kinase subunits, IKKalfa and IKKbeta, and a regulatory subunit IKKgamma (NEMO). Activation of IKK complex leads to phosphorylation of NF-κB inhibitor (IkB) and its degradation, resulting finally in the release of NF-κB and its translocation to the nucleus. AP-1

(Activator protein-1) is another transcription factor activated by this cascade.