Acute Phase Proteins

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Acute Phase Proteins David Samols1, Alok Agrawal1 and Irving Kushner2,* 1

Department of Biochemistry, Case Western Reserve University, School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106-4935, USA 2

Department of Medicine, MetroHealth Campus, Case Western Reserve University, 2500 MetroHealth Drive, Cleveland, OH 44109, USA * corresponding author tel: (216) 778-4765, fax: (216) 778-8376, e-mail: [email protected] DOI: 10.1006/rwcy.2002.0213.

INTRODUCTION The concentrations of many plasma proteins increase during inflammatory states, largely in response to inflammation-associated cytokines. C-reactive protein (CRP), the first such protein to be recognized, was initially detected in serum obtained from patients during the acute phase of pneumococcal pneumonia, hence the term `acute phase proteins' (APP). APPs are commonly defined as plasma proteins whose concentrations increase (positive acute phase proteins) by at least 25% during inflammatory states. In addition, a number of negative acute phase proteins, whose concentrations decrease significantly under these circumstances, have been recognized. All of these changes largely reflect altered production by hepatocytes. Maximal increases vary from about 50% in the case of ceruloplasmin and several complement components to over 1000-fold for CRP and serum amyloid A (SAA), the plasma precursor of amyloid A (the major constituent of secondary amyloid deposits). The best-studied human acute phase proteins are tabulated in Table 1. APPs are generally comparable in different mammalian species, with several noteworthy exceptions. For example, CRP, normally a trace protein in humans, rabbits, and mice, is massively induced (increases of a 1000-fold or more) by potent inflammatory stimuli in humans and rabbits, but is only minimally induced in mice. In contrast, CRP is constitutively expressed at relatively high levels in rats, with only a several-fold increase following stimulus. Other examples are 2-macroglobulin (2M), a major acute phase protein in the rat but

Cytokine Reference

not in humans, haptoglobin, a modest acute phase reactant in humans, but a major acute phase reactant in ruminants (in which it is normally undetectable) (Sheffield et al., 1994), and serum amyloid P, an acute phase reactant in mice but not in humans (Pepys et al., 1979). There are occasional interspecies problems in terminology, exemplified by the revision of SAA nomenclature in 1999 (Sipe, 1999). SAA is a family of genes consisting of three genes and a pseudogene in humans and four genes in mice. SAA1 and SAA2 are major acute phase genes in humans, while SAA4 is only modestly induced in inflammation and SAA3 is a pseudogene. In mice, Saa1, Saa2, and Saa3 are acute phase genes while Saa4 is a modest acute phase reactant and a pseudogene is designated SAA-psl. Acute phase plasma protein changes occur in the context of a very large number of other changes, distant from inflammatory sites and occurring in many organ systems, that are detectable within hours after the onset of an inflammatory response (Table 2) (Gabay and Kushner, 1999; Kushner, 1982), and can be regarded as the acute phase response (APR) in a broad sense. An APR comparable to that in humans is seen in all vertebrates, while lower species manifest lesser responses. At times there is an interrelationship between elements of the broad, systemic APR and individual APP. For example, a large number of changes in lipid metabolism occur during inflammatory states (Khovidhunkit et al., 2000), one of which is substantial replacement of the ApoA1 ordinarily found in association with the HDL3 fraction of lipoproteins by SAA (Coetzee et al., 1986).

Copyright # 2002 Published by Elsevier Science Ltd

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David Samols, Alok Agrawal and Irving Kushner Table 1

Some human acute phase proteins

Proteins whose plasma concentrations increase

Proteins whose plasma concentrations decrease

Complement system

Albumin

C3

Transferrin

C4

Transthyretin

C9

2 HS glycoprotein

Factor B

-Fetoprotein

C-1 inhibitor

Thyroxine-binding globulin

C4b-binding protein

Factor XII

Mannan-binding lectin

Retinol-binding protein

Participants in inflammatory responses Lipopolysaccharide-binding protein Granulocyte colony-stimulating factor IL-1 receptor antagonist Secretory phospholipase A2 Coagulation and fibrinolytic systems Fibrinogen Plasminogen Tissue plasminogen activator Plasminogen activator inhibitor- Urokinase Protein-S Vitronectin Antiproteases 1-Protease inhibitor 1-Antichymotrypsin Pancreatic secretory trypsin inhibitor Inter--trypsin inhibitors Transport proteins Ceruloplasmin Haptoglobin Hemopexin Others C-reactive protein Serum amyloid A 1-acid glycoprotein Fibronectin Ferritin Angiotensinogen

Protein C

Acute Phase Proteins 3 Table 2 Some components of the acute phase response Neuroendocrine changes Fever, somnolence, and anorexia Increased secretion of CRH, corticotropin, cortisol Increased secretion of arginine vasopressin Decreased insulin-like growth factor 1 production Increased adrenal secretion of catecholamines Hematopoietic changes Anemia of chronic disease Leukocytosis Thrombocytosis Metabolic changes Loss of muscle and negative nitrogen balance Decreased gluconeogenesis Osteoporosis Increased hepatic lipogenesis Increased lipolysis in adipose tissue Decreased lipoprotein lipase activity in muscle and adipose tissue Cachexia Hepatic changes Altered synthesis of plasma proteins Increased metallothionein, inducible nitric oxide synthase, heme oxygenase, manganese superoxide dismutase, tissue inhibitor of metalloproteinase-1 Decreased phosphoenolpyruvate carboxykinase activity Changes in nonprotein plasma constituents Decreased copper, iron, selenium, and zinc concentrations Decreased plasma retinol concentration Increased glutathione concentration

The APR is a major pathophysiologic phenomenon, in which normal homeostatic mechanisms are replaced by new set points that presumably contribute to defensive or adaptive capabilities. This latter likelihood has led to the realization that many of the changes occurring during the APR are part of the innate immune response (Medzhitov and Janeway, 2000; Munford and Pugin, 2001). Several semantic problems are associated with the APR. The use of this historically based term occasionally leads to confusion; the APR is not limited to acute inflammatory states, but may accompany chronic inflammation as well, resulting

in what might seem to be an oxymoron, a chronic APR. Another potential source of confusion is the use of the term `systemic inflammatory response' to refer to the APR. The systemic inflammatory response syndrome (SIRS) has already been defined as the earliest stage in the continuum of progressive decline that may occur following severe trauma or sepsis. It is characterized by high temperature, pulse rate, respiratory rate, and white cell counts, only one of which is regarded as part of the acute phase response (American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference, 1992). Finally, a number of investigators have employed the term `stress response' to refer to the neuroendocrine aspects of the APR (Chrousos, 1995). Many of these investigators have focused on the changes induced by psychological and emotional stimuli, often mediated by corticotropin-releasing hormone and the autonomic nervous system (Sternberg, 1997). These types of stimuli may also influence other, nonneuroendocrine components of the APR. Conditions that commonly lead to an APR include infection, trauma, surgery, burns, tissue infarction, various immunologically mediated and crystalinduced inflammatory conditions, and advanced cancer. Relatively modest changes occur after strenuous exercise, heatstroke, and childbirth. Recent studies have demonstrated that minimal acute phase protein elevations may be associated with a large number of conditions not ordinarily regarded as inflammatory, as discussed below under Clinical usefulness. Although multiple components of the acute phase response often occur together, not all of them occur uniformly in all patients with varying illnesses. Thus, discordance between plasma concentrations of different acute phase proteins is common. These variations, which indicate that components of the acute phase response are individually regulated, may be explained in part by differences in the patterns of production of specific cytokines (or their modulators) in different pathophysiologic states.

REGULATION OF GENE EXPRESSION As expected, the same general mechanisms that govern the regulation of gene expression elsewhere apply to acute phase protein genes (Brivanlou and Darnell, 2002). A series of regulatory elements are bound by transcription factors, which, associated with coactivators and corepressors, influence chromatin structure, the general transcriptional machinery

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and RNA polymerase II. However, in-depth studies of the molecular mechanisms that mediate the effects of cytokines on expression of individual AAP genes, as do in-depth studies of many genes, continue to reveal novel mechanisms by which gene regulation is accomplished. Examples, described in detail below, include a mechanism by which rel p50 enhances C/EBP functional effects on the CRP gene in the absence of p65, an effect of steroids on fibrinogen induction in the absence of a GRE, a requirement for STAT3 in the response of acute phase SAA to LPS or IL-6 in the absence of a defined STAT response element and displacement mechanisms whereby one transcription factor is replaced by another at an overlapping site.

Inducers of acute phase gene expression Although a wide variety of cytokines, growth factors, hormones, and other mediators have been found to influence the expression of acute phase protein genes in liver or liver-derived cells in culture, the major stimuli for induction of acute phase changes are the inflammation-associated cytokines. These molecules contribute to the inflammatory process at inflammatory sites largely by paracrine or autocrine mechanisms, while induction of APP changes results from bloodborne effects upon hepatocytes. Of these cytokines, members of the IL-6 family, TNF, IL-1, TGF , and IFN have been studied in some detail. IL-6 is the major stimulator of most acute phase proteins (Gauldie et al., 1987), while the other implicated cytokines influence subsets of acute phase proteins. The effects of these cytokines are influenced by circulating modulators of cytokine function, such as IL-1 receptor antagonist (IL-1Ra) and soluble TNF receptors, which inhibit the effects of their cognate cytokines, and by soluble IL-6 receptors, which enhance the effects of IL-6. In addition, other inflammation-associated cytokines such as IL-4 and IL-10, which have general antiinflammatory effects, inhibit induction of some acute phase proteins (Loyer et al., 1993; Vasse et al., 1996; Gabay et al., 1999). Finally, other circulating mediators such as HGF, insulin, EGF, CSFs, and glucocorticoids may influence the response to cytokines. While mediators capable of induction could theoretically alter gene expression by translational, posttranscriptional, or transcriptional mechanisms, it is transcriptional changes that are thought to account for most acute phase protein changes (Birch and

Schreiber, 1986), while translational mechanisms have not been found (Schreiber et al., 1986). Posttranscriptional mechanisms may participate as well, as has been shown for SAA and several other APPs (Jiang et al., 1995; Longley et al., 1999). Indeed, some cytokines, notably IL-1, have been shown to influence the stability of mRNAs in several systems (Winzen et al., 1999; Holtmann et al., 2001).

C/EBP and rel protein effects on APP gene expression The transcription factors that mediate enhanced expression of acute phase proteins in response to IL6, IL-1, and TNF typically include C/EBP family members (C/EBP and C/EBP), STAT proteins (STAT1, STAT3 and STAT5), rel family members (typically NFB), HNF1, Sp1 and AP-1. Regulatory elements identified in early studies of acute phase gene promoter regions as acute phase or IL-6 response elements have since been defined. Thus, the original consensus `IL-6RE sequence' (IL-6RE), is now recognized as a C/EBP-binding motif (Koj, 1996), while the subsequently defined `acute phase response element' (APRE), is a binding site for STAT proteins (Wegenka et al., 1994; Ripperger et al., 1995). Baumann and Gauldie (1990) proposed that the major acute phase genes be divided into two classes, depending on whether or not they responded to IL-1, in addition to responding to IL-6. Although probably not precisely accurate in detail, as each acute phase gene responds to a specific set of stimuli employing a unique combination of regulatory elements and transcription factors, this classification has provided a useful framework in which to analyze APP responses to cytokines. Class I (IL-1-responsive) genes include the SAA genes, CRP, 1-acid glycoprotein (AGP), C3, and rat haptoglobin. The promoters of all these genes contain binding sites for members of the C/EBP and rel families, often in close proximity (Poli, 1998). While all members of the C/EBP family bind to similar sequences (Lamb and McKnight, 1991; Osada et al., 1996), it is C/EBP and C/EBP isoforms of this family that are usually activated in association with inflammatory events. One or both of the C/EBP- or NFB-binding sites (called B sites) appear to be critical for full induction of class I genes in response to IL-6 and IL-1 (Prowse and Baumann, 1989; Wilson et al., 1990; Betts et al., 1993; Cha-Molstad et al., 2000). IL-1 is known to activate rel proteins and C/EBP (Isshiki et al., 1990), among others. IL-6 can activate C/EBP and can induce new synthesis of C/EBP (Alam et al., 1992; Cantwell et al., 1998).

Acute Phase Proteins 5 Interaction between rel and C/EBP families of transcription factors may account, in part, for the synergy often observed between IL-1 and IL-6 in this class of genes (Xia et al., 1997). In the best-studied example, the acute phase SAA genes, both IL-6 and IL-1 alone modestly induce expression, but their combination is markedly synergistic. This can be attributed to activation of C/EBP by IL-6, activation of NFB by IL-1 and physical interactions between RelA (p65) and C/EBP , since the binding sites for these transcription factors are closely spaced (Li and Liao, 1992; Betts et al., 1993; Ray et al., 1995; Xia et al., 1997). In contrast, in Hep3B cells, IL-1 alone has no effect on CRP expression, but markedly enhances CRP induction by IL-6. Studies of CRP induction in this cell line reveal that rel proteins also participate, but in a novel way; an interaction between C/EBP and rel p50 in a region where their binding sites overlap in the proximal promoter appears to be critical. For IL-1 effects to be evident, IL-6-induced transcription factor activation appears to be a prerequisite and the C/EBP-binding sites in the promoter region indispensable (Cha-Molstad et al., 2000; Agrawal et al., 2001). Participation of C/EBP family members, responsive to both IL-6 and IL-1, in acute phase protein induction is of particular interest. C/EBP is generally downregulated during the acute phase response (Alam et al., 1992) and C/EBP-containing dimers on acute phase gene promoters are often replaced by those composed of C/EBP and C/EBP (Poli, 1998). However, mice with a targeted disruption in the C/EBP gene do not manifest an APR and do not activate STAT3 in response to LPS. These data are consistent with a model in which C/EBP participates indirectly in the APR (BurgessBeusse and Darlington, 1998). C/EBP and C/EBP mRNA accumulation are increased by cytokine exposure in the liver (Takiguchi, 1998). In addition, existing C/EBP is activated by cytokines to translocate from the cytosol to the nucleus. There is conflicting evidence on the role of phosphorylation of C/EBP and C/EBP as a mechanism for their activation (Roesler, 2001). In hepatocytes, phosphorylation of C/EBP following TNF exposure is associated with its translocation from the cytoplasm to the nucleus. On the other hand, in vitro phosphorylated C/EBP does not bind DNA as well as its unphosphorylated counterpart (Trautwein et al., 1994). In contrast, phosphorylation has been associated with both translocation and stronger DNA binding of C/EBP, after IL-1 treatment, resulting in repression of the apoliprotein C-III promoter (Lacorte et al., 1997).

STAT protein effects on APP gene expression A number of class II acute phase genes, specifically the three chains of fibrinogen, 1-protease inhibitor, rat thiostatin, and rat 2M, are much more dependent on STAT3 activation than are class I genes. These genes often have multiple STAT-binding sites that are critical for their induction, and may lack C/EBPbinding sites. Since IL-6, but not IL-1, is the major activator of STAT3, it is not surprising that these genes are not induced by IL-1. In contrast to many acute phase genes, IL-1 represses basal and IL-6induced transcription of the fibrinogen genes, perhaps because many putative B sites overlap STAT3binding sites (APREs) (Zhang and Fuller, 2000). Such overlapping sites permit competition between NFB and STAT3; activated NFB may act as a repressor of fibrinogen transcription by displacing STAT3 (Fuller and Zhang, 2001). Of course, STAT3-binding sites can also be found in the promoters of class I genes such as CRP (Zhang et al., 1996), where they mediate part of the IL-6 response. In this model, STAT3 cooperates with C/EBP and rel proteins to affect full induction by IL-6 + IL-1. Although a STAT3-binding site has not been reported in the acute phase SAA gene promoters, mice in which the STAT3 gene was ablated in liver, adipose tissue, and bone marrow show impaired induction of (class 1) SAA1 and SAA2, as well as other acute phase genes, in response to LPS (Alonzi et al., 2001). Further, ablation of STAT3 , the dominant-negative splice variant of STAT3, led to overexpression of many acute phase genes, again indicating that STAT3 is an important regulator of most if not all acute phase proteins (Yoo et al., 2002).

Synergistic induction of APP gene expression More than additive responses, synergy, in cytokineinduced APP gene expression, is most likely due to transcription factor interactions. Most of the known examples of synergy involve rel protein interactions with other transcription factors. This is the case for the acute phase SAA genes (Betts et al., 1993; Shimizu and Yamamoto, 1994; Xia et al., 1997), CRP (Cha-Molstad et al., 2000; Agrawal et al., 2001), and C3 (Wilson et al., 1990). For fibrinogen (Fuller and Zhang, 2001) and AGP (Klein et al., 1987), synergy involves interactions with the glucocorticoid receptor (GR). STAT3 acts synergistically with GR (Zhang et al., 1997) on the fibrinogen- promoter while STAT3 and

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c-Jun (AP-1) interact with it on the 2M promoter (Yoo et al., 2001). All of these interactions probably function through cooperative coactivator binding, although this has been demonstrated thus far only for the coactivator TIF-1 binding to C/EBP and GR (Chang et al., 1998). Other transcription factor interactions include those between C/EBP and Sp1, and between C/EBP and Myb (Takiguchi, 1998). Finally, STAT3 and AP-1 induce expression of HNF1 (Leu et al., 2001) which is a necessary, constitutively active transcription factor that facilitates and is required for the expression of many acute phase and other liver genes. Thus, part of the synergistic responses to cytokines of some acute phase genes may reflect changes in HNF1 levels. Glucocorticoids are generally positive effectors of acute phase gene expression in hepatocytes, although their effects are minimal in the absence of cytokines. Glucocorticoid action can be direct or indirect. Some acute phase genes contain well-defined glucocorticoid response elements (GRE) in their promoters. Examples include AGP (Baumann and Maquat, 1986), fibrinogen- and fibrinogen- (Fuller and Zhang, 2001). In others, glucocorticoids apparently act indirectly and provide general support. In the case of the fibrinogen- gene, which has no recognizable GREs in its promoter sequence, it has been proposed that the glucocorticoid receptor interacts with STAT3 either by physical interaction or through a coactivator when STAT3 is bound to an APRE (Zhang et al., 1997; Fuller and Zhang, 2001). In addition to the well-characterized transcription factors described above, less well-characterized factors have been shown to participate in acute phase gene regulation. A factor designated SAF has been described which participates in regulation of acute phase SAA genes. It also regulates fibrinogen- through interactions with the ubiquitous transcription factor Sp1 (Ray and Ray, 1997; Ray, 2000). A mitochondrial DNA-binding protein, P16, has been reported to bind to a cis-element in the fibrinogen- promoter (Liu et al., 1997) and a constitutive factor, SAA3 enhancing factor (SEF), has been shown to participate in murine SAA3 expression. It has been suggested that SEF affects the promoters of other acute phase genes as well (Bing et al., 1999).

Negative and extra-hepatic regulation of APP gene expression Repression of acute phase protein gene expression has also been reported, particularly at extra-hepatic

sites. Rat SAA1 gene expression in HeLa cells is repressed by a displacement mechanism in which NFB and YY1 compete for overlapping binding sites. If YY1 is present, NFB is displaced from its B site, repressing expression from the gene (Lu et al., 1994; Li and Liao, 1999). Following cytokine exposure, YY1 is presumably either not expressed or expressed at low levels in hepatocytes. Similarly, AP-2 binds at two sites in the rat SAA1 promoter and can also repress transcription by displacing NFB (Ren and Liao, 2001). As described above (Zhang and Fuller, 1997), competition between STAT3 and NFB for binding to a critical IL-6response element in the fibrinogen- promoter explains the inhibitory effect of IL-1 on fibrinogen expression. While C/EBP, NFB, and AP-2 are known to activate transcription, as indicated above, they can all also act as repressors, depending on the context. Despite the presence of repression mechanisms, extra-hepatic synthesis of acute phase protein genes is abundantly documented (Aldred et al., 1992). Often these genes are regulated by different mechanisms than those acting in hepatocytes. Thus, in contrast to hepatocytes, brain expression of SAA1 is not responsive to IL-1 + IL-6 (Tucker and Sack, 2001). Aortic smooth muscle cell expression of the acute phase SAA genes does not occur in the absence of glucocorticoids (Kumon et al., 2001) and synovial cell expression of SAA1, unlike hepatocytes, involves cooperation between SP1 and SAF (Ray et al., 1999). In intestinal expression of AGP, it is TGF , rather than IL-1 or IL-6, that induces C/EBP isoforms (Yoo et al., 2001). Activation of Src and STAT3 has been shown to participate in CRP expression in fibroblasts (Turkson et al., 1998). CRP expression has been reported in several other tissues outside the liver, including neurons (Yasojima et al., 2000), atherosclerotic plaques (Yasojima et al., 2001), lacrymal glands (Wei et al., 2001), and mononuclear cells (Murphy et al., 1991). The mechanisms underlying CRP expression in these sites are as yet unexplained.

FUNCTIONS OF ACUTE PHASE PROTEINS Discussion of the function of the APR must be tempered by several caveats. First, we presume that acute phase changes play a beneficial role in adaptation and defense ± that they are part of the innate immune response ± because we are inclined to believe that nature tries to accomplish useful

Acute Phase Proteins 7 purposes. This is not necessarily true. The host response may be either protective or detrimental; maladaptive sequelae of the inflammatory response such as the SIRS, described above, multiple organ failure and death may occur (Dhainaut et al., 2001; Marshall, 2001). Second, our presumptions about the functions of acute phase proteins are largely based on their known functional capabilities in vitro and on logical speculation as to how these may serve useful purposes. We are not always certain that these presumptions are valid in in vivo situations. Finally, we must bear in mind that many of these molecules are multifunctional and may participate in the innate immune response, or in adapting to it, in more than one way, including ways we are not yet aware of. With these reservations in mind, it is appropriate to adduce functional roles for many acute phase proteins, either in enhancing or in restraining some of the complex cascades and interactions that occur in inflammatory states (Kushner, 1998), or in adapting to the perturbations in metabolism that occur during these states. APPs are felt to participate in a variety of inflammation-related activities, such as killing or limiting dispersion of pathogens, protecting the host from destructive elements of the inflammatory response (e.g. inhibition of proteases) and repair of tissue damage. Some APPs are directly harmful to microbes, while others target sites for cellular responses. Some work alone while others participate in cascades. An overview of the probable roles of APP can be gained by citing several examples, beginning with CRP, SAA, and AGP. CRP is a good example of why simple classification of acute phase proteins as either pro- or antiinflammatory may not be valid. A very large number of binding specificities and biologic effects of CRP have been reported (Mortensen, 2001; Volanakis, 2001). A major function of CRP is presumed to be `proinflammatory', related to its ability to specifically bind to phosphocholine and to some nuclear components. It can thus recognize some foreign pathogens as well as both phospholipid and nuclear constituents of damaged or necrotic cells. Further, when bound to one of its ligands, CRP can activate the complement system by the classical pathway. CRP may also interact with Fc receptors on phagocytic cells and act as an opsonin (Bharadwaj et al., 1999; Hundt et al., 2001). These findings suggest that CRP plays a proinflammatory role by initiating elimination of targeted bacteria or cells by interaction with humoral and cellular effector systems of inflammation. Indeed, protection from lethal bacterial infection has been observed in transgenic mice expressing CRP (Volanakis, 2001; Szalai, 2002). A proinflammatory role for CRP is further supported

by the observation that CRP can induce production of inflammatory cytokines (Ballou and Lozanski, 1992) and tissue factor (Cermak et al., 1993) by monocytes, as well as inducing shedding of IL-6 receptors by polymorphonuclear leukocytes (Jones et al., 1999). In addition, CRP has been found to induce adhesion molecule expression in endothelial cells in the presence of serum (Pasceri et al., 2000) and to bind to degraded low-density lipoprotein with subsequent complement activation (Bhakdi et al., 1999). In contrast, studies of the effects of CRP on chemotaxis, phagocytosis, and respiratory burst activity of phagocytic cells suggest a significant antiinflammatory role. CRP has been reported to inhibit neutrophil chemotaxis, to inhibit superoxide generation by neutrophils (Foldes-Filep et al., 1992; Shephard et al., 1992), and to induce large amounts of IL-1Ra in peripheral blood mononuclear cells (Tilg and Peschel, 1996). Anti-inflammatory effects could result from the ability of CRP to cause enhanced shedding of L-selectin (Zouki et al., 1997). Recently, CRP has been shown to bind to apoptotic lymphocytes, with consequent anti-inflammatory effects (Gershov et al., 2000). CRP can bind factor H, with consequent inhibition of the alternative complement pathway and a diminished inflammatory response (Jarva et al., 1999). Finally, studies in transgenic mice, in which CRP is protective against complement-induced alveolitis (Webster et al., 1994) and endotoxemia (Xia and Samols, 1997) suggest that the net effect of CRP in vivo is anti-inflammatory (Ahmed et al., 1996). Taken together, these data suggest that CRP may play multiple roles in the course of inflammatory processes. The primary physiological function of SAA, the other major human acute phase protein, similarly remains elusive. SAA has been shown to induce adhesion and chemotaxis of phagocytic cells and lymphocytes (Xu et al., 1995) acting through formyl peptide receptors (Su et al., 1999; Le et al., 2001). It is also reported to induce cytokine production (Patel et al., 1998) and metalloproteinase secretion (Migita et al., 1998). In addition, the findings that macrophages bear specific binding sites for SAA and that SAA-rich high-density lipoproteins display increased ability to transfer cholesterol to macrophages at inflammatory sites suggest a role of SAA in transfer of cholesterol to inflammatory cells (Lindhorst et al., 1997; Artl et al., 2000). SAA has also been reported to enhance low-density lipoprotein oxidation in arterial cell walls (Berliner et al., 1995). Finally, SAA3 produced by rabbit synovial fibroblasts induces synthesis of collagenase (Mitchell et al., 1991).

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A number of protective and anti-inflammatory effects of AGP have been reported. AGP binds to bacterial endotoxin and protects mice from endotoxin-induced septic and hypovolemic shock (Moore et al., 1997; Muchitsch et al., 1998). Transgenic mice overexpressing AGP are protected from Klebsiella pneumoniae infection, suggesting a role in nonspecific resistance to infection (Hochepied et al., 2000). Antiinflammatory effects of AGP include inhibition of neutrophil activation and modulation of lymphocyte responsiveness (Williams et al., 1997). AGP interacts with plasminogen activator inhibitor type 1 and stabilizes its inhibitory activity (Boncela et al., 2001). Finally, AGP exerts anti-apoptotic and anti-inflammatory effects in ischemia/reperfusion injury and some murine models of TNF toxicity (Van Molle et al., 1999; Daemen et al., 2000).

Complement system members Complement components, most of which have been found to be APPs, are long-recognized opsonins and mediators of the inflammatory response (Cooper, 1999). Activation of the complement system results in generation of three anaphylotoxins, C3a, C4a, and C5a, which can induce vascular permeability and smooth muscle contraction in blood vessels and lead to release of histamine and other vasoactive substances. C5a is also a chemotaxin and induces phagocytic cells to secrete inflammatory mediators, aggregate, and adhere to surfaces. Finally, activation of terminal complement components generates the membrane attack complex, a series of protein±protein interactions involving C5±C9, with lysis of target pathogens. Mannan-binding lectin (MBL) is an APP that initiates one of the three independent complement activation pathways and is an important component of innate immunity (Petersen et al., 2001). It binds to a wide range of pathogenic bacteria, viruses, fungi, and parasites as a result of its predefined lectin specificity, with consequent initiation of complement activation through complexed MBL-associated proteases.

Participants in inflammatory responses Lipopolysaccharide (LPS)-binding protein (LBP), an important participant in innate immunity, facilitates interaction between LPS and its receptor CD14 on phagocytic cells (Hailman et al., 1994; Schumann

et al., 1996). The lipid A portion of LPS binds to LBP in plasma and is delivered to the cell surface receptor CD14, where it is transferred to toll-like receptor 4 (TLR4), capable of transmembrane signaling through its accessory protein MD2. Rapid activation of an intracellular signaling network (similar to the signaling systems of IL-1 and IL-18) ensues, with resultant induction of many genes encoding inflammatory mediators such as cytokines, adhesion molecules, and tissue factor, as well as to activation of neutrophils (Alexander and Rietschel, 2001; Guha and Mackman, 2001). Non-CD14-bearing cells, such as endothelial cells, can also respond to LPS through this pathway via the soluble form of CD14, an interaction that is similarly enhanced by LBP. It has been proposed that LBP can also facilitate neutralization of LPS under certain conditions (Lamping et al., 1998). Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of hematopoietic cells and is the principal growth factor regulating maturation, proliferation, and differentiation of neutrophil precursors (Aritomi et al., 1999). In addition to this obviously proinflammatory function, G-CSF can stimulate superoxide production (Lindemann et al., 1991) and expression of Fc- RI (Kerst et al., 1993) in neutrophils. G-CSF phosphorylates a Cu/Zn-SOD through receptor-associated kinases, diminishes Cu/Zn-SOD levels and activity (Csar et al., 2001) and enhances antibody-dependent cytotoxicity (Stockmeyer et al., 2001). In contrast, IL1Ra is an APP of hepatic origin that binds avidly to IL-1 receptors with no apparent agonist function, thus inhibiting the effects of IL-1. It is presumed that the functional role played by this, and the many other `anti-inflammatory' molecules that are produced early in the course of inflammatory states (Gabay et al., 1997b; Tanuma et al., 1997), is to modulate the effects of potentially deleterious proinflammatory processes.

Coagulation system It is not surprising that a number of proteins of the coagulation and fibrinolytic systems are APPs, in light of the phylogenetically ancient association between coagulation and inflammation; invertebrates possess a `common cellular and humoral pathway of inflammation and clotting' in response to trauma or infection (Opal, 2000). In recent years, there has been increasing recognition that molecules involved in regulation of hemostasis also influence inflammatory pathways (Esmon, 2001; van der Poll, 2001). Fibrinogen is an excellent example: a multifunctional glycoprotein that plays important, overlapping roles

Acute Phase Proteins 9 in hemostasis, inflammation, and wound healing (Clark, 2001; Mosesson et al., 2001). In addition to participating in formation of clots, it stimulates secretion of chemokines by macrophages (Smiley et al., 2001). Treatment of neutrophils with fibrinogen in the presence of formyl-methionyl-leucyl-phenylalanine or leukotriene B4 results in increased IL-8 synthesis (Kuhns et al., 2001). Binding of fibrinogen to leukocyte integrins has been shown (Ugarova and Yakubenko, 2001) and it has been reported that fibrinogen is an important antioxidant (Kaplan et al., 2001).

Antiproteases and transport proteins Both antiproteases and the transport proteins (which manifest antioxidant activity) can be presumed to protect the host against potentially harmful components of the inflammatory response. Inflammation involves both release of proteases from phagocytic cells and activation of serum proteases, as well as generation of potentially toxic reactive oxygen species. Thus, 1-proteinase inhibitor (1-PI) (also called 1-antitrypsin) can inactivate a large number of serine proteases, but its major function appears to be to inhibit neutrophil elastase. In addition, it can inhibit neutrophil cathepsin G and proteinase 3, as well as mast cell proteinase II (Stockley, 2001). The critical role played by of some of these proteases in destruction of bacteria within neutrophils has recently been established (Reeves et al., 2002). It stands to reason that acute phase antiproteases serve to limit the activity of these powerful enzymes when they escape to the extracellular milieu. Like AGP, 1-PI has been reported to exert anti-apoptotic and antiinflammatory effects in murine models of ischemia/ reperfusion injury and in a model of TNF/ galactosamine toxicity (Van Molle et al., 1999; Daemen et al., 2000). Finally, 1-PI inhibits neutrophil superoxide production and induces macrophage-derived IL-1Ra (Bucurenci et al., 1992; Tilg et al., 1993). Similarly, 1-antichymotrypsin (1-ACT) strongly inhibits neutrophil cathepsin G and mast cell chymase. An additional anti-inflammatory role is suggested by the finding that 1-ACT inhibits the activity of the macrophage cell surface enzyme that facilitates conversion of pro-macrophage-stimulating protein (pro-MSP) to active MSP (Skeel and Leonard, 2001). Both 1-PI and 1-ACT prevent degradation of extracellular matrix proteins caused by cell-derived proteinases, permitting cell attachment and spreading (Ikari et al., 2001). Another serpin, C-1 inhibitor (listed in Table 1 under complement system)

not only participates in control of the classic complement activation pathway, but also inhibits kallikrein, an enzyme that contributes to inflammation by generating bradykinin and by amplifying the intrinsic pathway of coagulation (Caliezi et al., 2000). Ceruloplasmin (Cp), originally described as a copper transport molecule, is a multifunctional enzyme and a multifunctional protein (Floris et al., 2000) which plays essential roles in both iron and copper metabolism and integrates these metabolic pathways (Mzhel'skaya, 2000). Its major role during the APR may be to act as an antioxidant, due largely to its ferroxidase activity. Cp plays an important role in mobilization and oxidation of iron from tissue stores, with subsequent incorporation of ferric iron into transferrin, thus preventing the generation of reactive oxygen species. In addition, it can inactivate reactive oxygen species such as superoxide and hydrogen peroxide (Halliwell and Gutteridge, 1990). Finally, Cp modulates the function of endothelial nitric oxide synthase and thus controls NO-dependent relaxation of the vasculature (Bianchini et al., 1999). Haptoglobin (Hp) binds circulating hemoglobin liberated as a result of intravascular hemolysis, with consequent clearance of the complexes thus formed by cellular receptors (Kristiansen et al., 2001). In this way, it inhibits free-hemoglobin-induced lipid peroxidation (Halliwell and Gutteridge, 1990) and protects kidneys from damage. Hp knockout mice are more sensitive than wild-type mice to phenylhydrazineinduced hemolysis, supporting its protective role during hemolysis (Lim et al., 2000). In addition, Hp binds to the -2 integrin Mac-1, as does fibrinogen, suggesting that these APPs might regulate Mac-1dependent cell function. (El Ghmati et al., 1996). Hemopexin (Hx) is also felt to prevent oxidative damage. It binds free heme, which like hemoglobin, is capable of producing peroxidation (Gutteridge and Smith, 1988). In addition, Hx strongly inhibits the stimulatory effect of heme on growth of Bacteroides fragilis, an anaerobic pathogen (Rocha et al., 2001), and activates a number of genes that encode proteins important for cellular defenses against oxidative stress, such as heme oxygenase-1 and the cysteinerich metallothioneins (Sung et al., 2000). In summary, the presumed functions of APPs affect many aspects of the innate immune response, and consequent inflammation, including cytokine responses, coagulation, inflammatory cell behavior, vascular alterations and complement activation. APPs may protect against degradative enzymes and oxidative processes and may contribute to wound healing. In addition, they may affect metabolic behavior during illness and ultimately, influence the subsequent adaptive immune response.

10 David Samols, Alok Agrawal and Irving Kushner

CLINICAL USEFULNESS Determination of serum (or plasma) concentrations of APPs is useful to clinicians, since they reflect the presence and intensity of inflammatory processes (Gabay and Kushner, 1999). Although they lack diagnostic specificity, levels of APPs are helpful in differentiating inflammatory from noninflammatory conditions and in guiding patient management during the course of inflammatory diseases. Finally, higher concentrations of APPs indicate a poor prognosis in a wide variety of conditions, including rheumatoid arthritis, various malignancies, diabetes mellitus, and uremia. Currently, the most widely used indicators of the APP response are the erythrocyte sedimentation rate (ESR) and serum CRP concentration. The rate at which erythrocytes fall through plasma, the ESR, generally reflects degree of inflammation. It is influenced by both known and unknown factors but correlates moderately well with plasma concentrations of the APP fibrinogen (Bedell and Bush, 1985). The ESR has the advantages of familiarity, simplicity, and an abundant literature compiled over nearly eight decades. However, ESR values are imprecise and sometimes misleading, since they are only an indirect measure of APP concentrations and can be greatly influenced by the size, shape, or number of erythrocytes, by other plasma constituents such as monoclonal immunoglobulins, by age and by unknown factors. In contrast, CRP levels reflect synthesis of a single acute phase protein, directly measured, whose concentration changes much more rapidly in response to worsening or improvement in the inflammatory process than does the ESR and which is only minimally influenced by the subject's age and sex (Wener et al., 2000). Multiple components of the APR commonly, but not invariably, occur together. Discordance between concentrations of different APPs in different diseases and different patients is not uncommon; some APPs may be elevated while others are not. For example, many patients with active systemic lupus erythematosus have elevated ESR, but normal CRP concentrations. These patients are, however, capable of mounting a CRP response, as shown by marked increases in CRP concentrations during bacterial infection. Such variations in APPs, which indicate that components of the APR are individually regulated, may be explained, at least in part, by differences in production of specific cytokines or their modulators in different diseases. Accordingly, it appears safe to say that there is no single best laboratory test to reflect inflammation. Many

clinicians measure several acute phase reactants, rather than employing a single test, and interpret them in light of the clinical context. Most normal subjects have CRP concentrations of 2 mg/L or less, but some have concentrations as high as 10 mg/L. The latter finding, long attributed to modest stimulation by minimally apparent, low-grade inflammatory processes such as gingivitis, or by trivial injury incurred in the course of daily living, has led to the suggestion that values should be over 10 mg/L to be regarded as clinically significant (Morley and Kushner, 1982; Macy et al., 1997). However, recent data indicate that the finding of CRP concentrations between 2 and 10 mg/L may have some clinical relevance. Most commonly employed laboratory assays for CRP are not sensitive in this range; a `high sensitivity' CRP assay is required (Conti, 2001). The interpretation of such a minimal APR has recently become a subject of considerable interest. Minimally elevated APPs, including CRP, are associated with the progression of atherosclerosis. They predict the risk of a first myocardial infarction among apparently healthy individuals and are associated with a worse prognosis among patients with stable and unstable angina (Haverkate et al., 1997; Ridker et al., 1997). In the elderly, elevated levels of acute phase proteins predict `failure to thrive' and even increased mortality (Harris et al., 1999). A possible explanation for this phenomenon is, of course, that these individuals have an ongoing inflammatory process, in coronary arteries or elsewhere. However, CRP values in this range are associated with a large number of states not ordinarily regarded as inflammatory. Among these conditions are obesity, poor physical conditioning, high protein diet, depression and notably high or low alcohol intake (Fleming, 2000; Geffken et al., 2001; Imhof et al., 2001; Kushner, 2001). It may be that minimally elevated acute phase protein levels merely identify individuals who bear an increased burden of tissue damage resulting from cumulative oxidative stress, a process strongly implicated in the pathogenesis of aging (Kushner, 2001). Such individuals are biologically (not necessarily chronologically) older and as such, would be expected to have a greater likelihood of disease or death. In addition, recent data indicate that baseline CRP levels are heritable and that a promoter polymorphism of the IL-6 gene (-174G/C) is associated with high CRP levels (Vickers et al., 2002). At present, it is far from clear that high sensitivity CRP screening to identify individuals at risk for atherosclerosis, as some suggest (Ridker et al., 2001), will prove to be an effective procedure (Campbell et al., 2002; Kushner, 2002).

Acute Phase Proteins Currently, other acute phase reactants are rarely employed clinically. SAA concentrations usually parallel those of CRP. Although SAA is probably a more sensitive marker of inflammatory disease (Malle and De Beer, 1996), assays for SAA are not widely available at present. In addition, a high degree of sensitivity is not always desirable in the clinical setting, because of attendant loss of specificity. While plasma concentrations of cytokines and cytokine receptors have been studied in patients with inflammatory conditions, quantitation of cytokines presents several problems, partly because of their short plasma half-lives and the presence of blocking factors (May et al., 1988; Arend et al., 1994). Reports of different patterns of cytokine responses in different diseases suggest that cytokine determinations may ultimately have diagnostic value (Gabay et al., 1994, 1997a), as does the report that IL-6 is more sensitive than ESR for indicating disease activity in giant cell arteritis (Weyand et al., 2000). However, until further studies are available, high cost, limited availability, and absence of standardization will retard measurement of plasma cytokines and their receptors in clinical practice.

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