**5. SAA receptors**

It has long been suspected that the diverse functions of SAA are mediated by cell surface receptors. Studies conducted in the past 20 years have led to the identification of several cell surface receptors for SAA in addition to a number of binding proteins (**Figure 2**). In 1999, Su et al. reported the involvement of formyl peptide receptor 2 (FPR2, also termed FPRL1 [72, 73]), in the chemotactic activity of SAA [74]. FPR2 is a G protein-coupled chemoattractant receptor initially identified as a homolog of human FPR1 with low-affinity binding of formylated peptide [75–77]. The identification of FPR2 as a receptor for SAA is consistent with reports that SAA induces migration of phagocytes and to a lesser extent, lymphocytes [54, 55]. Subsequent studies have shown that a number of biological functions of SAA, ranging from chemotaxis and superoxide generation to induced expression of proinflammatory cytokines and matrix metalloproteases, are mediated through FPR2 [47, 60, 78–85].

**117**

*Serum Amyloid A and Immunomodulation DOI: http://dx.doi.org/10.5772/intechopen.81617*

**Figure 2.**

upon exposure to environmental stress.

The identification of cytokine-like activities of recombinant SAA protein suggests the involvement of receptors that typically mediate phagocyte cytokine production. The finding that SAA selectively induces IL-23 but not IL-12 expression suggests a pattern similar to that of Toll-like receptor (TLR)-mediated cytokine induction [65]. In 2008, two of the TLRs were identified as SAA receptors. TLR2, and more specifically the TLR2-TLR1 heterodimer, was found to mediate SAAinduced NF-κB activation leading to the expression of several proinflammatory cytokines and chemokines [86]. TLR2 is also responsible for SAA-induced neutrophil expansion through upregulation of G-CSF [64]. TLR4 was found to mediate SAA-induced expression of iNOS and activation of the related signaling pathways [87]. Despite differences in primary and high-level structures between SAA and the microbial ligands for these receptors, the two TLRs mediate SAA functions both in transfected cells expressing the receptors and *in vivo* [48, 71, 79, 88–92]. The identification of the two TLRs as SAA receptors illustrates the possible roles for TLRs in detecting host-derived molecules as a mechanism for alerting immune cells

*SAA receptors. The major receptors of human and mouse SAA proteins and their projected functions are listed. Also shown in the figure are selected binding partners of SAA. OmpA, bacterial outer membrane protein a; HDL, high-density lipoprotein. Permission from the publisher was obtained for the use of the crystal structure of SAA1 [32].*

RAGE (receptor for advanced glycation end product) is a multiligand immunoglobulin superfamily cell surface molecule. In a study of AA amyloidosis, RAGE was identified as a receptor of SAA [93]. The expression of RAGE and its interaction with SAA coincide with cell stress, and RAGE has been shown to mediate the NF-κB activating effect of SAA [93, 94]. SAA also binds to soluble RAGE [63]. NF-κB activation induced by SAA interaction with RAGE apparently contributed to the expression of tissue factor in monocytes through MAP kinase activation. Inhibition of RAGE by a RAGE competitor, by soluble RAGE, and by anti-RAGE IgG reduced the SAAstimulated tissue factor expression [63]. RAGE is also reported to mediate the proinflammatory activity of SAA in uremia-related atherosclerosis, based on a study using the *Apoe*<sup>−</sup>/<sup>−</sup> and *Ager*<sup>−</sup>/<sup>−</sup> mice [95]. These studies identify RAGE as an endothelial and

monocyte-expressed molecule that mediates selected activities of SAA.

Scavenger receptors on macrophages play important roles in the removal of debris during tissue injury and in macrophage transport of lipids. The scavengereceptor SR-BI has been known for mediating cholesterol efflux, in which SAA plays a role [96]. Two independent studies published in the same year reported the identification of SR-BI as an SAA receptor [97, 98]. Direct binding assays using radiolabeled SAA found its interaction with SR-B1 in cells that express this receptor [97]. SR-BI and its human homolog CLA-I mediate SAA uptake and its downstream signaling, including the activation of ERK and p38 MAPK that leads to IL-8

#### *Serum Amyloid A and Immunomodulation DOI: http://dx.doi.org/10.5772/intechopen.81617*

**Figure 2.**

*Amyloid Diseases*

production and neutrophil expansion [64]. SAA also induced the expression of immunomodulatory cytokines including selective induction of IL-23 over IL-12 [65] and the induction of IL-33 expression [66]. The transcription factors NF-κB, IRF4 and IRF7 have been implicated in SAA-induced gene expression [66, 67]. In addition,

SAA appears to be involved in epigenetic regulation of gene expression [68].

function of SAA in the induced expression of IL-1β.

It has long been suspected that the diverse functions of SAA are mediated by cell surface receptors. Studies conducted in the past 20 years have led to the identification of several cell surface receptors for SAA in addition to a number of binding proteins (**Figure 2**). In 1999, Su et al. reported the involvement of formyl peptide receptor 2 (FPR2, also termed FPRL1 [72, 73]), in the chemotactic activity of SAA [74]. FPR2 is a G protein-coupled chemoattractant receptor initially identified as a homolog of human FPR1 with low-affinity binding of formylated peptide [75–77]. The identification of FPR2 as a receptor for SAA is consistent with reports that SAA induces migration of phagocytes and to a lesser extent, lymphocytes [54, 55]. Subsequent studies have shown that a number of biological functions of SAA, ranging from chemotaxis and superoxide generation to induced expression of proinflammatory cytokines and

matrix metalloproteases, are mediated through FPR2 [47, 60, 78–85].

One of the cellular targets of SAA is macrophages, a major source of cytokines and most if not all SAA receptors. Macrophages may be differentiated into M1 or M2 phenotypes. Studies have shown that SAA may influence macrophage differentiation. Anthony et al. examined the effects of SAA *in vitro*, using human blood monocytes from chronic obstructive pulmonary disease patients and healthy controls, and *in vivo* using a mouse model with airway SAA challenge [69]. Their work showed that SAA-rendered human monocytes secrete IL-6 and IL-1β concurrently with the M2 markers CD163 and IL-10. Moreover, these cells responded to subsequent LPS stimulation with markedly higher levels of IL-6 and IL-1β. In the mouse model, SAA induced a CD11chigh CD11bhigh macrophage population in a CSF-1R signaling-dependent manner, with concurrent inhibition of neutrophilic inflammation. Sun et al. investigated the potential effect of SAA on macrophage plasticity, and found that SAA treatment led to increased expression of macrophage M2 markers including IL-10, Ym1, Fizz-1, MRC1, IL-1Rn, and CCL17 [67]. Moreover, SAA enhanced efferocytosis of mouse macrophages. Silencing IRF4 by small interfering RNA abrogated the SAA-induced expression of M2 markers, suggesting a potential role for SAA to alter macrophage phenotype and modulate macrophage functions. SAA has been identified as an endogenous activator of the NLRP3 inflammasome, which is critical to the process of pro-IL-1β. Niemi et al. reported that SAA provided a signal for pro-IL-1β expression and for inflammasome activation [70]. At least 3 SAA receptors, including TLR2, TLR4 and the ATP receptor P2X7, were involved. Interestingly, inflammasome activation was dependent on the activity of cathepsin B, the expression of which was induced by SAA. Therefore, SAA-induced secretion of cathepsin B could facilitate extracellular processing of SAA and development of AA amyloidosis. Ather et al. showed SAA3 expression in the lungs of mice exposed to mixed Th2/Th17-polarizing allergic sensitization regimens [71]. SAA instillation into the lungs elicited pulmonary neutrophilic inflammation and activation of the NLRP3 inflammasome, thereby promoting IL-1β secretion by dendritic cells and macrophages. SAA administered into the lungs also served as an adjuvant that sensitized mice to inhaled OVA, promoting IL-17 production from restimulated splenocytes and leukocyte influx. Collectively, these findings illustrate a stimulatory

**116**

**5. SAA receptors**

*SAA receptors. The major receptors of human and mouse SAA proteins and their projected functions are listed. Also shown in the figure are selected binding partners of SAA. OmpA, bacterial outer membrane protein a; HDL, high-density lipoprotein. Permission from the publisher was obtained for the use of the crystal structure of SAA1 [32].*

The identification of cytokine-like activities of recombinant SAA protein suggests the involvement of receptors that typically mediate phagocyte cytokine production. The finding that SAA selectively induces IL-23 but not IL-12 expression suggests a pattern similar to that of Toll-like receptor (TLR)-mediated cytokine induction [65]. In 2008, two of the TLRs were identified as SAA receptors. TLR2, and more specifically the TLR2-TLR1 heterodimer, was found to mediate SAAinduced NF-κB activation leading to the expression of several proinflammatory cytokines and chemokines [86]. TLR2 is also responsible for SAA-induced neutrophil expansion through upregulation of G-CSF [64]. TLR4 was found to mediate SAA-induced expression of iNOS and activation of the related signaling pathways [87]. Despite differences in primary and high-level structures between SAA and the microbial ligands for these receptors, the two TLRs mediate SAA functions both in transfected cells expressing the receptors and *in vivo* [48, 71, 79, 88–92]. The identification of the two TLRs as SAA receptors illustrates the possible roles for TLRs in detecting host-derived molecules as a mechanism for alerting immune cells upon exposure to environmental stress.

RAGE (receptor for advanced glycation end product) is a multiligand immunoglobulin superfamily cell surface molecule. In a study of AA amyloidosis, RAGE was identified as a receptor of SAA [93]. The expression of RAGE and its interaction with SAA coincide with cell stress, and RAGE has been shown to mediate the NF-κB activating effect of SAA [93, 94]. SAA also binds to soluble RAGE [63]. NF-κB activation induced by SAA interaction with RAGE apparently contributed to the expression of tissue factor in monocytes through MAP kinase activation. Inhibition of RAGE by a RAGE competitor, by soluble RAGE, and by anti-RAGE IgG reduced the SAAstimulated tissue factor expression [63]. RAGE is also reported to mediate the proinflammatory activity of SAA in uremia-related atherosclerosis, based on a study using the *Apoe*<sup>−</sup>/<sup>−</sup> and *Ager*<sup>−</sup>/<sup>−</sup> mice [95]. These studies identify RAGE as an endothelial and monocyte-expressed molecule that mediates selected activities of SAA.

Scavenger receptors on macrophages play important roles in the removal of debris during tissue injury and in macrophage transport of lipids. The scavengereceptor SR-BI has been known for mediating cholesterol efflux, in which SAA plays a role [96]. Two independent studies published in the same year reported the identification of SR-BI as an SAA receptor [97, 98]. Direct binding assays using radiolabeled SAA found its interaction with SR-B1 in cells that express this receptor [97]. SR-BI and its human homolog CLA-I mediate SAA uptake and its downstream signaling, including the activation of ERK and p38 MAPK that leads to IL-8

expression [98]. A more recent study reported that SR-BII, a splice variant of SR-BI, also serves as a SAA receptor for uptake and proinflammatory signaling through MAP kinase signaling [99].

The human P2X7 purinergic receptor is an ionotropic receptor found at high expression levels in immune cells such as macrophages and microglia. Activation of P2X7 receptor by extracellular ATP opens a cation channel, allowing K<sup>+</sup> efflux that is associated with processing of pro-interleukin IL-1β and IL-18. Christenson et al. found that SAA, either recombinant or purified from the plasma of rheumatoid arthritis patients, could suppress apoptosis of human neutrophils, an effect abrogated by antagonizing the nucleotide receptor P2X7 [100]. Niemi et al. reported that the P2X7 receptor plays a role in SAA-mediated activation of NLRP3, thereby explaining the involvement of SAA in the processing of pro-IL-1β [70]. However, a recently published work indicates that in murine J774 and bone marrow-derived macrophages, SAA stimulates IL-1β secretion through a mechanism that depends on NLRP3 expression and caspase-1 activity but not the P2X7 receptor [101].

Collectively, published reports have identified several functional receptors that mediate SAA signaling. It is likely that these receptors and their downstream signaling pathways have substantial cross-talk that together contributes to the diverse immunomodulatory and homeostatic functions of SAA.

Recent studies have shown that recombinant human SAA, which has been widely used in in *vitro* studies throughout the last two decades, has properties that differ from those of native SAA purified from human samples [102–104]. The rhSAA differs from human SAA1 in two sites, with amino acid substitutions at positions 60 and 71 in addition to gaining a methionine at the N-terminus. Since the rhSAA is made by *Escherichia coli* expression, the bacterial contaminants in the preparation may contribute to the observed cytokine-like activity. This is especially a concern because the contaminating bacterial products can activate the two TLRs that are known as the SAA receptors. A careful analysis of published literature found evidence that both support the use of the two TLRs by SAA and detract from the claim. Many of the published studies have included controls for LPS contamination, showing that the SAA protein is necessary for the reported biological functions. A recent study has shown that the bacterial contaminants may not be LPS that acts through TLR4 but lipoproteins that activate TLR2 [105]. The study also showed that adding bacterial lipopeptides into mammalian cell-expressed SAA1 protein could restore the cytokine-like activity that otherwise was missing from the SAA1 protein [105]. It is however unclear how much lipoproteins are carried by the *E. coli*-derived recombinant SAA. The *E. coli* expression system has been widely used in the production of reagents including proinflammatory cytokines such as TNFα and IL-1β, and there were not previous concerns over bacterial product contamination with these cytokines. Whereas the authors attributed the previously reported NLRP3 inflammasome-activating property of SAA to bacterial lipoprotein contaminants in the *E. coli*-derived SAA [105], another recent study demonstrated that SAA purified from human samples was able to stimulate NLRP3 inflammasome activation [101]. Taken together, these findings raise the possibility that bacterial contaminants may modify the biological properties of human SAA1 for a potent cytokine-inducing effect. Exactly how much bacterial contaminant is associated with a recombinant human SAA1 is still unknown, but published studies have shown that *E. coli*-produced SAA proteins can be processed to sufficient purity so they can form crystals [32, 56]. Moreover, CHO cell-derived SAA in the form of secreted Fc fusion protein has been shown to bind to the ectodomain of TLR2 [86]. While the contaminating lipoproteins may contribute to the cytokine-inducing activity through TLR2, these contaminants have not been known to stimulate the G protein-coupled FPR2 that mediates some of the biological activities of SAA [47, 60, 74, 78–85]. Based on available data, it is postulated that some of the observed functions of rhSAA are

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physiologically relevant conditions.

role for SAA3 in atherosclerosis [108].

attributable to bacterial contaminants. *In vivo* studies conducted in various models of diseases are therefore important for confirming the biological functions of SAA under

Since most of the early studies were conducted using cell lines and isolated primary cells such as monocytes and neutrophils, these experimental findings are now examined in an *in vivo* setting. An early model created for the *in vivo* studies of SAA employed adenoviral expression of human SAA1, raising the circulatory levels of human SAA1 in the infected mice [106]. This approach was used in studies of the involvement of SAA1 in lipid metabolism [106] and fibril formation [107]. In a more recent study, the same group that created the adenoviral approach found a

Transgenic expression of human SAA1 in mice is another approach used in studies of the *in vivo* functions of SAA. Ji et al. reported transgenic expression of human SAA1 in mouse liver [89]. These mice exhibited more severe liver injury, increased hepatocyte apoptosis, and higher levels of hepatic enzymes than in their

and macrophages was also increased more in the transgenic mice than in wildtype mice, along with elevated expression of several chemokines. The aggravated liver injury, increased hepatocyte apoptosis and elevated levels of hepatic enzymes in the transgenic mice were eased with the use of a TLR2 antagonist, suggesting that TLR2 mediates the effects of the transgenic SAA1. In a more recent study, Cheng et al. placed the human SAA1 under an inducible promoter of SR-A receptor, generating transgenic mice with elevated local production of SAA1 upon inflammatory stimulation [53]. The transgenic SAA1 was most abundant in mouse lungs and protected mice against acute lung injury caused by LPS administration and cecal ligation and puncture (CLP). Transgenic expression of SAA1 did not protect mice against acute lung injury induced by intratracheal instillation of TNFα. Binding studies showed that human SAA1, purified from either *E. coli* or transfected HEK293 cells, bound to LPS and formed a complex that promoted LPS clearance by macrophages. As a result, serum endotoxin concentration was significantly reduced in the transgenic mice than in their wildtype controls that went through the CLP procedure. Of note, injection of a SAA1-derived peptide that disrupted LPS-SAA1 interaction diminished the endotoxin-lowering effect in the SAA1 transgenic mice and increased serum endotoxin level in wildtype mice after CLP [53]. These findings suggest a mechanism by which

T cells

wildtype controls. After induction of hepatitis, liver infiltration of CD4+

acute-phase SAA protects host against bacterial infection-induced injury.

SAA gene knockout mice were generate to examine the physiological functions of the individual SAA proteins. After observing SAA1 and SAA2 expression in intestinal epithelial cells and conforming their cell-protecting effect in epithelial cell line co-cultured with *E. coli*, Eckhardt et al. examined the effect of Saa1/2 double knockout (DKO) in dextran sodium sulfate (DSS) induced colitis model [109]. They found that that epithelial expression of SAA1 and SAA2 protected colonic epithelium against bacterial infection. A more recent study using Saa3 gene knockout mice found that SAA3 is the predominant isoform of inducible SAA proteins in colonic epithelium following chemical injury [92]. Compared to wildtype mice, *Saa3*<sup>−</sup>/<sup>−</sup> mice exposed to DSS showed more severe damage to the colonic epithelial structure, significantly reduced expression of the anti-microbial peptides Reg3β and Reg3γ, and reduced lifespan of afflicted mice if not treated. Administration of exogenous SAA3 protein or adoptive transfer of SAA3-treated neutrophils partially ameliorated symptoms of DSS-induced colitis in part due to SAA3-induced

**6. Immunomodulatory functions of SAA in disease models**

*Amyloid Diseases*

MAP kinase signaling [99].

expression [98]. A more recent study reported that SR-BII, a splice variant of SR-BI, also serves as a SAA receptor for uptake and proinflammatory signaling through

The human P2X7 purinergic receptor is an ionotropic receptor found at high expression levels in immune cells such as macrophages and microglia. Activation of

is associated with processing of pro-interleukin IL-1β and IL-18. Christenson et al. found that SAA, either recombinant or purified from the plasma of rheumatoid arthritis patients, could suppress apoptosis of human neutrophils, an effect abrogated by antagonizing the nucleotide receptor P2X7 [100]. Niemi et al. reported that the P2X7 receptor plays a role in SAA-mediated activation of NLRP3, thereby explaining the involvement of SAA in the processing of pro-IL-1β [70]. However, a recently published work indicates that in murine J774 and bone marrow-derived macrophages, SAA stimulates IL-1β secretion through a mechanism that depends on

Collectively, published reports have identified several functional receptors that mediate SAA signaling. It is likely that these receptors and their downstream signaling pathways have substantial cross-talk that together contributes to the diverse

Recent studies have shown that recombinant human SAA, which has been widely used in in *vitro* studies throughout the last two decades, has properties that differ from those of native SAA purified from human samples [102–104]. The rhSAA differs from human SAA1 in two sites, with amino acid substitutions at positions 60 and 71 in addition to gaining a methionine at the N-terminus. Since the rhSAA is made by *Escherichia coli* expression, the bacterial contaminants in the preparation may contribute to the observed cytokine-like activity. This is especially a concern because the contaminating bacterial products can activate the two TLRs that are known as the SAA receptors. A careful analysis of published literature found evidence that both support the use of the two TLRs by SAA and detract from the claim. Many of the published studies have included controls for LPS contamination, showing that the SAA protein is necessary for the reported biological functions. A recent study has shown that the bacterial contaminants may not be LPS that acts through TLR4 but lipoproteins that activate TLR2 [105]. The study also showed that adding bacterial lipopeptides into mammalian cell-expressed SAA1 protein could restore the cytokine-like activity that otherwise was missing from the SAA1 protein [105]. It is however unclear how much lipoproteins are carried by the *E. coli*-derived recombinant SAA. The *E. coli* expression system has been widely used in the production of reagents including proinflammatory cytokines such as TNFα and IL-1β, and there were not previous concerns over bacterial product contamination with these cytokines. Whereas the authors attributed the previously reported NLRP3 inflammasome-activating property of SAA to bacterial lipoprotein contaminants in the *E. coli*-derived SAA [105], another recent study demonstrated that SAA purified from human samples was able to stimulate NLRP3 inflammasome activation [101]. Taken together, these findings raise the possibility that bacterial contaminants may modify the biological properties of human SAA1 for a potent cytokine-inducing effect. Exactly how much bacterial contaminant is associated with a recombinant human SAA1 is still unknown, but published studies have shown that *E. coli*-produced SAA proteins can be processed to sufficient purity so they can form crystals [32, 56]. Moreover, CHO cell-derived SAA in the form of secreted Fc fusion protein has been shown to bind to the ectodomain of TLR2 [86]. While the contaminating lipoproteins may contribute to the cytokine-inducing activity through TLR2, these contaminants have not been known to stimulate the G protein-coupled FPR2 that mediates some of the biological activities of SAA [47, 60, 74, 78–85]. Based on available data, it is postulated that some of the observed functions of rhSAA are

efflux that

P2X7 receptor by extracellular ATP opens a cation channel, allowing K<sup>+</sup>

NLRP3 expression and caspase-1 activity but not the P2X7 receptor [101].

immunomodulatory and homeostatic functions of SAA.

**118**

attributable to bacterial contaminants. *In vivo* studies conducted in various models of diseases are therefore important for confirming the biological functions of SAA under physiologically relevant conditions.
