also produced by liver cells.

addition (Wolk et al., 2004).

skin, and liver.

**5. IL-22 expression and functions**

proteins, mainly SAA, fibrinogen, CRP that are indicative of an APR.

of Acute Phase Proteins and Antimicrobial Peptides as Players of Homeostasis 93

constitutive production of blood proteins and one of the major sources of acute-phase proteins. Thus, liver in uncontrolled local tissue inflammation processes produce, IL-6 and other family members, TNF-α, and IL-1β leading the production of these acute-phase

APR participate at a very early stage after stimulus by recognizing non-self elements in the body, **quickly after infection**, preceding both the complete activation and the setting up of immune responses. Also, the acute-phase response is resolved as soon as stimulation disappears. These APR regulation is due to the expression of inflammation regulators including IL-10, SOCS, soluble receptors of inflammatory cytokines (IL-1ra) as well as due to

Activated immune cells including monocytes and lymphocytes release pro-inflammatory cytokines to stimulate hepatocytes; subsequently induce synthesis and secretion of acute phase proteins. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and interleukin-6 (IL-6) following their pattern-recognition receptors (PRRs) bind pathogen associated molecular patterns (PAMPs). IL-22 is a more recently discovered cytokine that is

A study on HepG2 hepatocellular carcinoma-derived cell line allowed to identify an IL-22– dependent induction of APP gene expression (Dumoutier et al., 2000). Wolk et al. subsequently reported increased levels of circulating SAA as a consequence of IL-22

Interleukin-22 (IL-22), a cytokine from the IL-10 family but exhibit low homology with IL-10, produced by several immune cell subsets, mainly including (IL-6, TNFa)-stimulated Th-22 that will produce the largest amounts (37 up to 65% of the total depending on the tissue) in the absence of IL-17 production, IL-12-stimulted or activated (CD3) Th-1/Tbet+ (35%), (IL-1b, IL-6, TGFb, IL-23)-stimulated Th-17/RORgt+ (10%) that in addition will produce IL-17A&F IL-21 and IL-28. Innate lymphocytes NK22/NKp44+ (Duhen et al., 2009; Eyerich et al., 2009; Trifari et al., 2009) and NK cells (Wolk & Sabat, 2006) and γδ T and LTi-like cells that are IL-22 producers (Zenewicz et al., 2008). Other T cell subset have been shown an IL-22 production such as the T8 /Tc22 in atopic dermatitis (Nograles et al., 2009) and in

It has been demonstrated that IL-22 does not act on immune cells as compared with other T or NK cells secreted cytokines. Direct effects of IL-22 are restricted to non-hematopoietic cells carrying IL-22 receptors that are expressed on the surface of only epithelial cells and some fibroblasts in a large variety of organs, including parenchymal tissue of the gut, lung,

The expression of the IL-22R1 chain determines cells and organs exhibiting an IL-22 target profile (Boniface et al., 2005). Thus, the main IL-22R1 and IL-10R2 expressers are epithelial and fibroblast cells (Wolk et al., 2004; Wolk et al., 2005) excluding to the immune monocytoid or lymphoid cells and they are carriers of both IL-22R and IL-10R2 (Kotenko,

Depending on local inflammatory processes in its targeted tissues, this cytokine is quickly over-expressed in its source cells and exhibit three main documented activities (Fig 4): (i) **antimicrobial** by inducing AMPs (SAA, A-antitrypsin, haptoglobin, hepcidin, S100A7, S100A8 S100A9, -Def-1 & 2, RegIII etc), (ii) **tissue-damage protection** *via* induction of

psoriasis where also Tc17-Il-17A production was associated (Res et al., 2010).

2002; Langer et al., 2004; Pestka et al., 2004; Sabat et al., 2007)

of adherence, apoptosis of viral- or bacteria-infected host cells, inhibition of fibrin clot lysis (to limit bacterial spread) and inhibition of proteases (to limit tissue injury). If inflammation becomes chronic AMP will participate in both T cell recruitment as well as enhancement of monocyte chemotaxis and macrophage response.

A rich literature has reported results directly or indirectly demonstrating the efficiency of AMP. Mice carrying a genetically engineered metalloproteinase deficiency showed an increased susceptibility to bacterial infection generating an absence of soluble -defensins to protect mice against bacteria due to the absence of their extracellular cleavage and activation (Wilson et al., 1999). In a mouse model of septicemia, LL-37 binds to and neutralizes LPS and protects against endotoxic shock (Bals et al., 1999). Histatins protect from periodontal disease by inhibiting the *B. gingivalis* protease (Nishikata et al., 1991).

Due the efficiency against microbes the use of AMP, some AMP have been extracted from species different of those where effects are needed. This is the case of dermaseptin (DS), a 34 amino acid residue cationic peptide isolated from *Phyllomedusa sauvagii* skin, has been incubated *in vitro* with promastigotes of *Leishmania mexicana*. Immunocytochemical, and electron microscopic observations have shown that the amphipathic peptide generates disrupt the lipid bilayer leading of the surface membrane and death of the parasite (Hernandez et al., 1992).

Hepcidin (gene name, *HAMP*), an IL-6-inducible acute phase protein that also exhibit antimicrobial properties, is the key negative regulator of iron metabolism. Liver is the primary source of *HAMP* synthesis, but it is also produced by other tissues such as kidney or heart and is found in body fluids such as urine or cerebrospinal fluid. Hepcidin is a stress-inducible peptide of the biliary epithelia. In the bile, hepcidin help to protect against bacterial infections (Strnad et al., 2011).

Studies in transgenic mice confirmed that **C-reactive protein** is protective against microbial pathogens thru its *in vitro* ability to bind microbes, activating the complement classical and alternative pathways as well as FcγRII (Szalai, 2002).

SAA is the major acute phase protein in man and most mammals. Recently, it was demonstrated that SAA binds to many Gram-negative bacteria including *Escherichia coli* and *Pseudomonas aeruginosa* through outer membrane protein A (OmpA) family members. At normal concentrations of SAA, the SAA-*E coli* opsonization increase, both the rate of neutrophils with bacteria as well as the number of bacteria. The amount of neutrophil reactive oxygen intermediate production and phagocytosis by both neutrophils and IL-10 and TNFaproducers macrophages are enhanced in a SAA-dependent manner (Shah et al., 2006).

CRP have both properties to induce proinflammatory cytokines and tissues factor and antiinflammatory for example inhibiting superoxide anions secretion by neutrophils as well as inducing IL-1ra synthesis by PBMC. Serum amyloid A consists rapidly bind to high-density lipoprotein and influence cholesterol metabolism during inflammatory states (Banka et al., 1995). SAA could contribute to the inflammatory state by inducing chemotaxis and adhesion (Malle & De Beer, 1996).

#### **4. Cytokines and acute phase response**

A complex network of intercellular signaling involving cytokines, cytokine modulators and hormones regulates the acute-phase response (APR). Inflammation-associated cytokines, produced by both cell in the inflammatory site circulating immune cells, induce changes in expression of APP by liver cells. Liver is one of the primary organs required for the constitutive production of blood proteins and one of the major sources of acute-phase proteins. Thus, liver in uncontrolled local tissue inflammation processes produce, IL-6 and other family members, TNF-α, and IL-1β leading the production of these acute-phase proteins, mainly SAA, fibrinogen, CRP that are indicative of an APR.

APR participate at a very early stage after stimulus by recognizing non-self elements in the body, **quickly after infection**, preceding both the complete activation and the setting up of immune responses. Also, the acute-phase response is resolved as soon as stimulation disappears. These APR regulation is due to the expression of inflammation regulators including IL-10, SOCS, soluble receptors of inflammatory cytokines (IL-1ra) as well as due to short span of APP and their mRNA half-lives.

Activated immune cells including monocytes and lymphocytes release pro-inflammatory cytokines to stimulate hepatocytes; subsequently induce synthesis and secretion of acute phase proteins. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1beta), and interleukin-6 (IL-6) following their pattern-recognition receptors (PRRs) bind pathogen associated molecular patterns (PAMPs). IL-22 is a more recently discovered cytokine that is also produced by liver cells.

### **5. IL-22 expression and functions**

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

of adherence, apoptosis of viral- or bacteria-infected host cells, inhibition of fibrin clot lysis (to limit bacterial spread) and inhibition of proteases (to limit tissue injury). If inflammation becomes chronic AMP will participate in both T cell recruitment as well as enhancement of

A rich literature has reported results directly or indirectly demonstrating the efficiency of AMP. Mice carrying a genetically engineered metalloproteinase deficiency showed an increased susceptibility to bacterial infection generating an absence of soluble -defensins to protect mice against bacteria due to the absence of their extracellular cleavage and activation (Wilson et al., 1999). In a mouse model of septicemia, LL-37 binds to and neutralizes LPS and protects against endotoxic shock (Bals et al., 1999). Histatins protect from periodontal

Due the efficiency against microbes the use of AMP, some AMP have been extracted from species different of those where effects are needed. This is the case of dermaseptin (DS), a 34 amino acid residue cationic peptide isolated from *Phyllomedusa sauvagii* skin, has been incubated *in vitro* with promastigotes of *Leishmania mexicana*. Immunocytochemical, and electron microscopic observations have shown that the amphipathic peptide generates disrupt the lipid bilayer leading of the surface membrane and death of the parasite

Hepcidin (gene name, *HAMP*), an IL-6-inducible acute phase protein that also exhibit antimicrobial properties, is the key negative regulator of iron metabolism. Liver is the primary source of *HAMP* synthesis, but it is also produced by other tissues such as kidney or heart and is found in body fluids such as urine or cerebrospinal fluid. Hepcidin is a stress-inducible peptide of the biliary epithelia. In the bile, hepcidin help to protect against

Studies in transgenic mice confirmed that **C-reactive protein** is protective against microbial pathogens thru its *in vitro* ability to bind microbes, activating the complement classical and

SAA is the major acute phase protein in man and most mammals. Recently, it was demonstrated that SAA binds to many Gram-negative bacteria including *Escherichia coli* and *Pseudomonas aeruginosa* through outer membrane protein A (OmpA) family members. At normal concentrations of SAA, the SAA-*E coli* opsonization increase, both the rate of neutrophils with bacteria as well as the number of bacteria. The amount of neutrophil reactive oxygen intermediate production and phagocytosis by both neutrophils and IL-10 and TNFa-

CRP have both properties to induce proinflammatory cytokines and tissues factor and antiinflammatory for example inhibiting superoxide anions secretion by neutrophils as well as inducing IL-1ra synthesis by PBMC. Serum amyloid A consists rapidly bind to high-density lipoprotein and influence cholesterol metabolism during inflammatory states (Banka et al., 1995). SAA could contribute to the inflammatory state by inducing chemotaxis and adhesion

A complex network of intercellular signaling involving cytokines, cytokine modulators and hormones regulates the acute-phase response (APR). Inflammation-associated cytokines, produced by both cell in the inflammatory site circulating immune cells, induce changes in expression of APP by liver cells. Liver is one of the primary organs required for the

producers macrophages are enhanced in a SAA-dependent manner (Shah et al., 2006).

monocyte chemotaxis and macrophage response.

(Hernandez et al., 1992).

(Malle & De Beer, 1996).

bacterial infections (Strnad et al., 2011).

alternative pathways as well as FcγRII (Szalai, 2002).

**4. Cytokines and acute phase response**

disease by inhibiting the *B. gingivalis* protease (Nishikata et al., 1991).

A study on HepG2 hepatocellular carcinoma-derived cell line allowed to identify an IL-22– dependent induction of APP gene expression (Dumoutier et al., 2000). Wolk et al. subsequently reported increased levels of circulating SAA as a consequence of IL-22 addition (Wolk et al., 2004).

Interleukin-22 (IL-22), a cytokine from the IL-10 family but exhibit low homology with IL-10, produced by several immune cell subsets, mainly including (IL-6, TNFa)-stimulated Th-22 that will produce the largest amounts (37 up to 65% of the total depending on the tissue) in the absence of IL-17 production, IL-12-stimulted or activated (CD3) Th-1/Tbet+ (35%), (IL-1b, IL-6, TGFb, IL-23)-stimulated Th-17/RORgt+ (10%) that in addition will produce IL-17A&F IL-21 and IL-28. Innate lymphocytes NK22/NKp44+ (Duhen et al., 2009; Eyerich et al., 2009; Trifari et al., 2009) and NK cells (Wolk & Sabat, 2006) and γδ T and LTi-like cells that are IL-22 producers (Zenewicz et al., 2008). Other T cell subset have been shown an IL-22 production such as the T8 /Tc22 in atopic dermatitis (Nograles et al., 2009) and in psoriasis where also Tc17-Il-17A production was associated (Res et al., 2010).

It has been demonstrated that IL-22 does not act on immune cells as compared with other T or NK cells secreted cytokines. Direct effects of IL-22 are restricted to non-hematopoietic cells carrying IL-22 receptors that are expressed on the surface of only epithelial cells and some fibroblasts in a large variety of organs, including parenchymal tissue of the gut, lung, skin, and liver.

The expression of the IL-22R1 chain determines cells and organs exhibiting an IL-22 target profile (Boniface et al., 2005). Thus, the main IL-22R1 and IL-10R2 expressers are epithelial and fibroblast cells (Wolk et al., 2004; Wolk et al., 2005) excluding to the immune monocytoid or lymphoid cells and they are carriers of both IL-22R and IL-10R2 (Kotenko, 2002; Langer et al., 2004; Pestka et al., 2004; Sabat et al., 2007)

Depending on local inflammatory processes in its targeted tissues, this cytokine is quickly over-expressed in its source cells and exhibit three main documented activities (Fig 4): (i) **antimicrobial** by inducing AMPs (SAA, A-antitrypsin, haptoglobin, hepcidin, S100A7, S100A8 S100A9, -Def-1 & 2, RegIII etc), (ii) **tissue-damage protection** *via* induction of

IL-22 Induces an Acute-Phase Response Associated to a Cohort

the recovery phase (Sugimoto et al., 2008).

longevity of these subjects.

of Acute Phase Proteins and Antimicrobial Peptides as Players of Homeostasis 95

done a study on mice infected with LCMV clone-13 that exhibit persistent high-level viremia and a dysfunctional immune response. Interleukin-7, a cytokine that is critical for immune development and homeostasis, was used here to promote immunity toward clone-13, enabling elucidation of the inhibitory pathways underlying impaired antiviral immune response. IL-7 downregulated the critical repressor of cytokine signaling, Socs3, resulting in amplified cytokine production, increased T cell effector function and numbers, and viral clearance. Additionally, IL-7 promoted production of IL-22 that abrogated liver pathology. Recently an improvement intestinal inflammation in a mouse model of ulcerative colitis by IL-22 was mediated by enhanced mucus production has been reported. A local gene delivery was used to inhibit IL-22 activity through overexpression of IL-22-binding protein. Treatment with IL-22-binding protein suppressed goblet cell restitution (reparation) during

Aging is characterized by a progressive alteration of **homeostatic** mechanisms modulated by environmental and genetic factors. It is associated with a pro-inflammatory status. Cytokine dysregulation is believed to play a key role in the proposed remodeling of the immune-inflammatory responses accompanying old age. Centenarians displayed significantly higher circulating IL-22 levels compared to control population (Basile et al., 2011). This pro-inflammatory condition is probably not only protective against infections, but also it contributes to a better predisposition to preserve homeostasis, thus promoting the

Once the IL-22 expression is set up, the biological activity of this cytokine will be mainly regulated by two factors: (i) the expression level of its appropriated receptor, IL-22R1 on its target epithelial cells (Li et al., 2004; Xie et al., 2000) and (ii) the expression level and

Liang et al assessed effects of IL-22 in mice (Liang et al., 2010). By utilizing adenoviralmediated delivery of IL-22 and systemic administration of IL-22 protein, we observed that IL-22 modulates factors involved in coagulation, including fibrinogen levels and platelet numbers, and cellular constituents of blood, such as neutrophil and RBC counts. Thus, IL-22 induces effects on systemic biochemical, cellular, and physiological parameters as well as thymic atrophy, body weight loss, and renal proximal tubule metabolic activity. These cellular and physiological parameters are indicative of a proper APR (Gabay & Kushner, 1999) in systemic inflammatory state. AdIL-22 mouse infection induces sustained high levels of circulating SAA and fibrinogen respectively on days 3, 7, and 14 and on Days 1 3, and 7 in blood. These observations demonstrated that exposure IL-22 for 2 wk resulted in the modulation of acute-phase proteins of the acute phase response (Liang et al., 2010). Following the interaction between IL-22 and its receptors, several observations report that IL-22 induces **APP, anti-microbial peptides gene expression**, cytokines, chemokines, and matrix metalloproteinases (Boniface et al., 2005), from **skin, gut, lung, or liver** epithelial **cells**. IL-22 modulates local inflammation, through **mucosal barrier integrity** and generating a protective inflammatory response against mucosal entry pathogens including viruses

localization of the soluble IL-22 binding protein (IL-22 BP) (Dumoutier et al., 2001).

(Misse et al., 2007)and extracellular bacteria (Aujla & Kolls, 2009; Zheng et al., 2008).

The majority of IL-22 effects are benefic or **protective** (gut, vagina, lungs, liver) for the host, but in some situations of chronic inflammation, it has been reported that this cytokine could have **pathogenic** effects (skin, joints) including in noninfectious inflammatory disease states.

**6. IL-22 induces expression of an acute-phase response**

APR (IL-6, TNFa, CXCL1, S100A7, S100A8 S100A9, -Def-1 & 2, IL-20, etc) and (iii) **tissue repair and remodeling** by enhancing expression of MMP-1,2 as well as Muc-1, 3 10, 13 and some survival genes (Bcl-2, Bcl-XL).

Thus, a localized production of IL-22 in the liver seems to promote hepatocyte survival and proliferation, but could primes the liver to be more susceptible to tumor development without significantly affecting liver inflammation (Park et al., 2011). Damaged cultured keratinocytes become repaired after IL-22-dependent remodeling (Eyerich et al., 2009). AAT that has been reported as a potential biomarker for hepatitis B in diagnosis (Tan et al., 2011). IL-22 contributes to regenerate tissues following liver surgery (Ren et al., 2010) or alcoolcirrhotic damages (Ki et al., 2010).

Fig. 4. Mucosal innate protection and tissue damage, the double game of IL-17 & IL-22 cytokines is determined by the local environment situation. Main IL-22 pathways playing a pivotal role in the protection of mucosal tissues against intracellular and extracellular infectious agents such as extracellular bacterias or sexually transmitted viruses. In vivo or ex vivo data these localized anti-infectious activities could involve several possible pathways including both different mode of action of anti-microbials and the infectious pathways used by pathogens. In addition IL-22 will play an important role in both tissue damage protection through APP induction and regeneration.

In the aim to understand the factors that impede immune responses to persistent viruses is essential in designing therapies for HIV infection, Pellegrini *et al* (Pellegrini et al., 2011) have

APR (IL-6, TNFa, CXCL1, S100A7, S100A8 S100A9, -Def-1 & 2, IL-20, etc) and (iii) **tissue repair and remodeling** by enhancing expression of MMP-1,2 as well as Muc-1, 3 10, 13 and

Thus, a localized production of IL-22 in the liver seems to promote hepatocyte survival and proliferation, but could primes the liver to be more susceptible to tumor development without significantly affecting liver inflammation (Park et al., 2011). Damaged cultured keratinocytes become repaired after IL-22-dependent remodeling (Eyerich et al., 2009). AAT that has been reported as a potential biomarker for hepatitis B in diagnosis (Tan et al., 2011). IL-22 contributes to regenerate tissues following liver surgery (Ren et al., 2010) or alcool-

Fig. 4. Mucosal innate protection and tissue damage, the double game of IL-17 & IL-22 cytokines is determined by the local environment situation. Main IL-22 pathways playing a pivotal role in the protection of mucosal tissues against intracellular and extracellular infectious agents such as extracellular bacterias or sexually transmitted viruses. In vivo or ex vivo data these localized anti-infectious activities could involve several possible pathways including both different mode of action of anti-microbials and the infectious pathways used by pathogens. In addition IL-22 will play an important role in both tissue damage protection

In the aim to understand the factors that impede immune responses to persistent viruses is essential in designing therapies for HIV infection, Pellegrini *et al* (Pellegrini et al., 2011) have

some survival genes (Bcl-2, Bcl-XL).

cirrhotic damages (Ki et al., 2010).

through APP induction and regeneration.

done a study on mice infected with LCMV clone-13 that exhibit persistent high-level viremia and a dysfunctional immune response. Interleukin-7, a cytokine that is critical for immune development and homeostasis, was used here to promote immunity toward clone-13, enabling elucidation of the inhibitory pathways underlying impaired antiviral immune response. IL-7 downregulated the critical repressor of cytokine signaling, Socs3, resulting in amplified cytokine production, increased T cell effector function and numbers, and viral clearance. Additionally, IL-7 promoted production of IL-22 that abrogated liver pathology.

Recently an improvement intestinal inflammation in a mouse model of ulcerative colitis by IL-22 was mediated by enhanced mucus production has been reported. A local gene delivery was used to inhibit IL-22 activity through overexpression of IL-22-binding protein. Treatment with IL-22-binding protein suppressed goblet cell restitution (reparation) during the recovery phase (Sugimoto et al., 2008).

Aging is characterized by a progressive alteration of **homeostatic** mechanisms modulated by environmental and genetic factors. It is associated with a pro-inflammatory status. Cytokine dysregulation is believed to play a key role in the proposed remodeling of the immune-inflammatory responses accompanying old age. Centenarians displayed significantly higher circulating IL-22 levels compared to control population (Basile et al., 2011). This pro-inflammatory condition is probably not only protective against infections, but also it contributes to a better predisposition to preserve homeostasis, thus promoting the longevity of these subjects.

Once the IL-22 expression is set up, the biological activity of this cytokine will be mainly regulated by two factors: (i) the expression level of its appropriated receptor, IL-22R1 on its target epithelial cells (Li et al., 2004; Xie et al., 2000) and (ii) the expression level and localization of the soluble IL-22 binding protein (IL-22 BP) (Dumoutier et al., 2001).

#### **6. IL-22 induces expression of an acute-phase response**

Liang et al assessed effects of IL-22 in mice (Liang et al., 2010). By utilizing adenoviralmediated delivery of IL-22 and systemic administration of IL-22 protein, we observed that IL-22 modulates factors involved in coagulation, including fibrinogen levels and platelet numbers, and cellular constituents of blood, such as neutrophil and RBC counts. Thus, IL-22 induces effects on systemic biochemical, cellular, and physiological parameters as well as thymic atrophy, body weight loss, and renal proximal tubule metabolic activity. These cellular and physiological parameters are indicative of a proper APR (Gabay & Kushner, 1999) in systemic inflammatory state. AdIL-22 mouse infection induces sustained high levels of circulating SAA and fibrinogen respectively on days 3, 7, and 14 and on Days 1 3, and 7 in blood. These observations demonstrated that exposure IL-22 for 2 wk resulted in the modulation of acute-phase proteins of the acute phase response (Liang et al., 2010).

Following the interaction between IL-22 and its receptors, several observations report that IL-22 induces **APP, anti-microbial peptides gene expression**, cytokines, chemokines, and matrix metalloproteinases (Boniface et al., 2005), from **skin, gut, lung, or liver** epithelial **cells**. IL-22 modulates local inflammation, through **mucosal barrier integrity** and generating a protective inflammatory response against mucosal entry pathogens including viruses (Misse et al., 2007)and extracellular bacteria (Aujla & Kolls, 2009; Zheng et al., 2008).

The majority of IL-22 effects are benefic or **protective** (gut, vagina, lungs, liver) for the host, but in some situations of chronic inflammation, it has been reported that this cytokine could have **pathogenic** effects (skin, joints) including in noninfectious inflammatory disease states.

IL-22 Induces an Acute-Phase Response Associated to a Cohort

inflammation and demyelination (Levillayer et al., 2007).

up to mucosal tissues (Castelletti et al., 2007).

viral dissemination.

within the range of their inhibitory activity (Kramer et al., 2010).

extendable to humans.

of Acute Phase Proteins and Antimicrobial Peptides as Players of Homeostasis 97

physiopathological observations in animal models do not allow to extract conclusions

IL-22 could synergize with other cytokines such as IL-7, a cytokine that is critical for immune development and homeostasis, to protect mouse organs from pathogenic effects of a LCMV (clone-13). Mice infected with have persistent high-level viremia and a dysfunctional immune response. IL-7 was used to promote immunity toward clone-13, enabling elucidation of the inhibitory pathways underlying impaired antiviral immune response. Mechanistically, IL-7 downregulated a critical repressor of cytokine signaling, Socs3, resulting in amplified cytokine production, increased T cell effector function and viral clearance. IL-7 enhanced thymic output to expand the naive T cell pool, including T cells that were not LCMV specific. Additionally, IL-7 promoted production of cytoprotective IL-22 that abrogated liver pathology (Pellegrini et al., 2011). This example is not far from the observations reporting that the IL-22 expression was increased in the presence in viral hepatitis C cases but didn't show in vitro models a direct effect on HCV (Dambacher et al., 2008). In contrast, IL-22 alone could be considered to treat Theiler's virus-induced encephalomyelitis, a mouse infection characterized in susceptible animals by chronic

Means to avoid the HIV mucosal entry is a crucial objectives to fight against this viral dissemination. The earliest responses to acute HIV-1 infection (AHI) will be due to the action of the mucosal epithelial cells, macrophages and DCs, NK cells as well as antimicrobial factors such as beta-defensins, activated complement. We have shown that natural constitutive higher IL-22 levels of transcripts and proteins are found in repeatedly HIV-1-exposed, uninfected individuals (ESN) as compared with "normal healthy individuals at an approximative ratio 3/1. These higher quantities of one of the major innate inducer protection systems permit an absence of infection in ESN. IL-22 induces A-SAA that, in turn, will agonistically bind to formyl peptide cell receptor (FPR) and therefore induce indirect down modulation of CCR5 in the same immature myeloid dendritic cells, which consequently will drastically reduce HIV-1 infection (Misse et al., 2007). In addition, IL-22-mediated antiviral effects include the stimulation of the production of beta-defensins 2 and 3 by ectocervix epithelial cells as well (F. Veas, M. Clerici, unpublished observations). Some of these individuals could exhibit other complementary HIV protection systems such as APOBEC, or anti-CCR5 IgA antibodies (Miyazawa et al., 2009), as well as the over expression of CCL28 in ESN that helps massive migration of anti-viral IgA secretory B cells

It has been recently reported that in HIV infected persons, an induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT), was observed in plasma coincident with viremia. Both A-SAA and AAT fragment have anti-viral activity *in vitro* and quantitation of their plasma levels indicated that circulating concentrations are likely to be

IL-22 is a potent inducer of AAT and MMP then mucosal or topic treatments could be beneficial to avoid the HIV mucosal entry and therefore can be crucial to fight against this

High systemic levels of IL-10, CRP and IL-22 in HIV-1C-infected Indian patients were associated with low viral replication *in vitro*. Whereas using healthy-donor PBMC *in vitro*, these isolates exhibited a high replication capacity.in *In vitro*, pretreatment of virus cultures

In rheumatoid arthritis, IL-22 play an inflammatory role, may result in bone damage (Geboes et al., 2009). Thus, IL-22 alone has been reported to be able to induce skin hyperplasia and epidermal wound healing (Boniface et al., 2005; Eyerich et al., 2009). The effect of IL-22 signaling will depend on the local cellular situation. Surprisingly, it has been shown *T. trichiura* colonization of the intestine may reduce symptomatic colitis by promoting goblet cell hyperplasia and mucus production through Th2 cytokines and IL-22 (Broadhurst et al., 2010).

#### **7. IL-22 induces antimicrobial responses is depending of the "niche" circumstances**

Zheng et al. reported that *in vivo* IL-22 induced the anti-microbial peptides **named the RegIII** proteins in intestinal epithelial cells. An intestinal infection *Citrobacter rodentium* in IL-22-deficient mice induced death (Zheng et al., 2008). Moreover IL-22 in intestine is differentially generated by a subset of NK cells, NKp46+(Satoh-Takayama et al., 2008). Aujla et al. reported that in a mouse model IL-22 upregulated the expression of lipocalin 2 in lung tissue and increased the transepithelial resistance to bacteria, and consequently, and here also the IL-22 neutralization during pulmonary *Klebsiella pneumoniae* infection death of these animals is observed (Aujla & Kolls, 2009). During pulmonary *K. pneumoniae* infection, it seems that both IL-22 and IL-17A cytokines produced by T cells were necessary for the host defense. Whereas for intestinal infection with *C. rodentium*, IL-22 was produced earlier than IL-17A and this latter was dispensable. Also, independently of IL-17, IL-22 can protect against *Salmonella enteritica* that induces systemic infections (Schulz et al., 2008), due to IL-22 increased levels upon intraperitoneal mice contamination (Siegemund et al., 2009). In contrast, multi-microbial sepsis can be avoided by inhibiting IL-22 with IL-22BP-Fc 4 h before sepsis induction leading to enhanced accumulation of neutrophils and mononuclear phagocytes and a reduced bacterial load at the site of infection. In addition, IL-22 blockade led to an enhanced bacterial clearance in liver and kidney and reduced kidney injury. These results imply an important proinflammatory role of IL-22 during septic peritonitis, contributing to bacterial spread and organ failure(Weber et al., 2007). IL-22 therefore, appears to play an important role in the regulation of inflammatory processes *in vivo*.

Analysis of Th1, Th2, and Th17 cytokine responses in cultured PBMCs from individuals of a cohort of subjects infected with *Leishmania donovani* having developed a visceral disease Kala Azar (KA) or having been protected against KA showed that IL-17 and IL-22 were strongly and independently associated with protection against KA. These results suggest that, along with Th1 cytokines, IL-17 and IL-22 play complementary roles in human protection against KA, and that a defect in Th17 induction may increase the risk of KA(Pitta et al., 2009). However mechanisms of protection in this case remain unclear.

Some clinical reports on *Candida albicans* state that IL-22 is associated to the presence of IL-22 (Liu et al., 2009), and some established correlations tend to show that IL-22 could play a role in controlling this infection (Puel et al., 2010). In animal models the role of IL-22 seems related to IL-17 environment (De Luca et al., 2010), IL-22 will show positive effects if IL-17RA is deficient but in an IL-17-independent manner. Another report from mouse model studies on *C. albicans* state that IL-17 and IL-23 but not IL-22 and IL-12 are required (Kagami et al., 2010).

These contradictory results must be taken into account, because would IL-17 and IL-22 certainly do not exhibit the same behavior in humans and in mouse, consequently,

In rheumatoid arthritis, IL-22 play an inflammatory role, may result in bone damage (Geboes et al., 2009). Thus, IL-22 alone has been reported to be able to induce skin hyperplasia and epidermal wound healing (Boniface et al., 2005; Eyerich et al., 2009). The effect of IL-22 signaling will depend on the local cellular situation. Surprisingly, it has been shown *T. trichiura* colonization of the intestine may reduce symptomatic colitis by promoting goblet cell hyperplasia and mucus production through Th2 cytokines and IL-22

Zheng et al. reported that *in vivo* IL-22 induced the anti-microbial peptides **named the RegIII** proteins in intestinal epithelial cells. An intestinal infection *Citrobacter rodentium* in IL-22-deficient mice induced death (Zheng et al., 2008). Moreover IL-22 in intestine is differentially generated by a subset of NK cells, NKp46+(Satoh-Takayama et al., 2008). Aujla et al. reported that in a mouse model IL-22 upregulated the expression of lipocalin 2 in lung tissue and increased the transepithelial resistance to bacteria, and consequently, and here also the IL-22 neutralization during pulmonary *Klebsiella pneumoniae* infection death of these animals is observed (Aujla & Kolls, 2009). During pulmonary *K. pneumoniae* infection, it seems that both IL-22 and IL-17A cytokines produced by T cells were necessary for the host defense. Whereas for intestinal infection with *C. rodentium*, IL-22 was produced earlier than IL-17A and this latter was dispensable. Also, independently of IL-17, IL-22 can protect against *Salmonella enteritica* that induces systemic infections (Schulz et al., 2008), due to IL-22 increased levels upon intraperitoneal mice contamination (Siegemund et al., 2009). In contrast, multi-microbial sepsis can be avoided by inhibiting IL-22 with IL-22BP-Fc 4 h before sepsis induction leading to enhanced accumulation of neutrophils and mononuclear phagocytes and a reduced bacterial load at the site of infection. In addition, IL-22 blockade led to an enhanced bacterial clearance in liver and kidney and reduced kidney injury. These results imply an important proinflammatory role of IL-22 during septic peritonitis, contributing to bacterial spread and organ failure(Weber et al., 2007). IL-22 therefore, appears to play an important role in the regulation of inflammatory processes *in vivo*. Analysis of Th1, Th2, and Th17 cytokine responses in cultured PBMCs from individuals of a cohort of subjects infected with *Leishmania donovani* having developed a visceral disease Kala Azar (KA) or having been protected against KA showed that IL-17 and IL-22 were strongly and independently associated with protection against KA. These results suggest that, along with Th1 cytokines, IL-17 and IL-22 play complementary roles in human protection against KA, and that a defect in Th17 induction may increase the risk of KA(Pitta

**7. IL-22 induces antimicrobial responses is depending of the "niche"**

et al., 2009). However mechanisms of protection in this case remain unclear.

Some clinical reports on *Candida albicans* state that IL-22 is associated to the presence of IL-22 (Liu et al., 2009), and some established correlations tend to show that IL-22 could play a role in controlling this infection (Puel et al., 2010). In animal models the role of IL-22 seems related to IL-17 environment (De Luca et al., 2010), IL-22 will show positive effects if IL-17RA is deficient but in an IL-17-independent manner. Another report from mouse model studies on *C. albicans* state that IL-17 and IL-23 but not IL-22 and IL-12 are required (Kagami

These contradictory results must be taken into account, because would IL-17 and IL-22 certainly do not exhibit the same behavior in humans and in mouse, consequently,

(Broadhurst et al., 2010).

**circumstances**

et al., 2010).

physiopathological observations in animal models do not allow to extract conclusions extendable to humans.

IL-22 could synergize with other cytokines such as IL-7, a cytokine that is critical for immune development and homeostasis, to protect mouse organs from pathogenic effects of a LCMV (clone-13). Mice infected with have persistent high-level viremia and a dysfunctional immune response. IL-7 was used to promote immunity toward clone-13, enabling elucidation of the inhibitory pathways underlying impaired antiviral immune response. Mechanistically, IL-7 downregulated a critical repressor of cytokine signaling, Socs3, resulting in amplified cytokine production, increased T cell effector function and viral clearance. IL-7 enhanced thymic output to expand the naive T cell pool, including T cells that were not LCMV specific. Additionally, IL-7 promoted production of cytoprotective IL-22 that abrogated liver pathology (Pellegrini et al., 2011). This example is not far from the observations reporting that the IL-22 expression was increased in the presence in viral hepatitis C cases but didn't show in vitro models a direct effect on HCV (Dambacher et al., 2008). In contrast, IL-22 alone could be considered to treat Theiler's virus-induced encephalomyelitis, a mouse infection characterized in susceptible animals by chronic inflammation and demyelination (Levillayer et al., 2007).

Means to avoid the HIV mucosal entry is a crucial objectives to fight against this viral dissemination. The earliest responses to acute HIV-1 infection (AHI) will be due to the action of the mucosal epithelial cells, macrophages and DCs, NK cells as well as antimicrobial factors such as beta-defensins, activated complement. We have shown that natural constitutive higher IL-22 levels of transcripts and proteins are found in repeatedly HIV-1-exposed, uninfected individuals (ESN) as compared with "normal healthy individuals at an approximative ratio 3/1. These higher quantities of one of the major innate inducer protection systems permit an absence of infection in ESN. IL-22 induces A-SAA that, in turn, will agonistically bind to formyl peptide cell receptor (FPR) and therefore induce indirect down modulation of CCR5 in the same immature myeloid dendritic cells, which consequently will drastically reduce HIV-1 infection (Misse et al., 2007). In addition, IL-22-mediated antiviral effects include the stimulation of the production of beta-defensins 2 and 3 by ectocervix epithelial cells as well (F. Veas, M. Clerici, unpublished observations). Some of these individuals could exhibit other complementary HIV protection systems such as APOBEC, or anti-CCR5 IgA antibodies (Miyazawa et al., 2009), as well as the over expression of CCL28 in ESN that helps massive migration of anti-viral IgA secretory B cells up to mucosal tissues (Castelletti et al., 2007).

It has been recently reported that in HIV infected persons, an induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT), was observed in plasma coincident with viremia. Both A-SAA and AAT fragment have anti-viral activity *in vitro* and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity (Kramer et al., 2010).

IL-22 is a potent inducer of AAT and MMP then mucosal or topic treatments could be beneficial to avoid the HIV mucosal entry and therefore can be crucial to fight against this viral dissemination.

High systemic levels of IL-10, CRP and IL-22 in HIV-1C-infected Indian patients were associated with low viral replication *in vitro*. Whereas using healthy-donor PBMC *in vitro*, these isolates exhibited a high replication capacity.in *In vitro*, pretreatment of virus cultures

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with IL-10 and CRP resulted in a significant reduction of virus production, whereas IL-22, which lacks action on immune cells appears to mediate its anti-HIV effect through interaction with both IL-10 and CRP, and its own protective effect on mucosal membranes (Arias et al., 2010).

Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.

#### **8. Conclusion**

APR has major consequences during a local inflammation process with systemic consequences to maintain the whole organism homeostasis. The continuous discovery of new mediators/receptors of this response allow to more precisely tunes the knowledge about subtle necessary equilibrium to understand diseases set up to propose new possible therapeutic approaches. The double game of inflammatory processes to destroy the non-self and repair the self clearly is not ease to delimitate and this knowledge is a tremendous and pivotal challenge for a well adapted personalized medicine.

Moreover, data are mostly harvested from animal models that do not reflect physiopathological patterns that are generated in humans with their own kinetics. Nevertheless, today animal models are absolutely necessary, but observations need to be improved with more adapted reagents particularly for the *in vivo* real time observations of the physiopathological evolution and conditions.

Another imperious need is to assess functional concentrations of mediators/receptors such as IL-22 that are present in healthy individuals resisting to diseases such as infections, in a way to measure over the time their concentrations and understand their signification in terms of their homeostatic values in relation with complex networks

#### **9. Acknowledgement**

We are indebted to support from the Institut de Recherche pour le Développement, the University Montpellier 1 and the Région Languedoc-Roussillon, France.

#### **10. References**

with IL-10 and CRP resulted in a significant reduction of virus production, whereas IL-22, which lacks action on immune cells appears to mediate its anti-HIV effect through interaction with both IL-10 and CRP, and its own protective effect on mucosal membranes

Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future

APR has major consequences during a local inflammation process with systemic consequences to maintain the whole organism homeostasis. The continuous discovery of new mediators/receptors of this response allow to more precisely tunes the knowledge about subtle necessary equilibrium to understand diseases set up to propose new possible therapeutic approaches. The double game of inflammatory processes to destroy the non-self and repair the self clearly is not ease to delimitate and this knowledge is a tremendous and

Moreover, data are mostly harvested from animal models that do not reflect physiopathological patterns that are generated in humans with their own kinetics. Nevertheless, today animal models are absolutely necessary, but observations need to be improved with more adapted reagents particularly for the *in vivo* real time observations of

Another imperious need is to assess functional concentrations of mediators/receptors such as IL-22 that are present in healthy individuals resisting to diseases such as infections, in a way to measure over the time their concentrations and understand their signification in

We are indebted to support from the Institut de Recherche pour le Développement, the

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**5**

Irina I. Patalakh

*Ukraine*

**Hemostatic Soluble Plasma**

**Proteins During Acute-Phase**

**Response and Chronic Inflammation**

Coagulation and inflammation are the interdependent processes attributed to the host defence response to injuries. A synchronized activity of both pathways represents an essential prerequisite for restitution of host homeostasis after ultimate disturbances of the latter. Crosstalk between coagulation and inflammation is considered to inherit from primitive coagulation systems similar to invertebrates. For instance, in Horseshoe "crabs" (*Limulus*) coagulogen, a clotting protein with the bactericidal function, and coagulationrelated serine proteases are both located in circulating hemolymph cells (hemocytes) and are capable of simultaneously protecting against injury, as well as to isolate pathogens within cysts (Tanaka et al., 2009). In humans, the complement system as a part of innate immunity remains closely related to the hemostasis system (Markiewski et al., 2007). The organizations of these two systems demonstrate several similarities of both structural and functional features. Both systems are organized into proteolytic cascades; serine proteases of the chymotrypsin family are the components of the latter. Proteases of each system are, in their molecular structure, glycoproteins, which have a highly conservative catalytic site composed of serine, histidine, and aspartic amino acid residues. These proteases exist in the form of inactive zymogens and are subsequently activated by upstream, active proteases. Proteins of the complement and hemostasis systems are mostly synthesized in the liver by hepatocytes, besides being additionally produced during an acute-phase response stimulated by common inflammatory mediators. The biological role of the complement system is mentioned here in order to note that the above-mentioned similarities represent a forcible argument in favour of the common origin of both immunity and hemostasis phenomena. The complement system has been described in detail recently (Castellheim et al., 2009; Markiewski et al., 2007) and will not be further discussed here. Though, it is worthwhile to mention that the blood hemostasis system in humans, hypothetically, has been evolved progressively from the archaic innate immunity organization, diverging to

certain narrow-specific pathways, which are responsible for the coagulation control.

Hemostasis in humans is organized as closely interrelated enzyme cascade systems: (i) fibrin clotting system (coagulation); (ii) multilevel system for preventing uncontrolled fibrin formation (anticoagulation); and (iii) system for limiting the amount of cross-linked fibrin (fibrinolysis). Together, coagulation, anticoagulation and fibrinolysis are associated into a

**1. Introduction**

*Palladin Institute of Biochemistry of the NAS of Ukraine, Kyiv*

### **Hemostatic Soluble Plasma Proteins During Acute-Phase Response and Chronic Inflammation**

Irina I. Patalakh *Palladin Institute of Biochemistry of the NAS of Ukraine, Kyiv Ukraine*

#### **1. Introduction**

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

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Modrusan, Z., Ghilardi, N., de Sauvage, F. J. & Ouyang, W. (2008). Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens. Coagulation and inflammation are the interdependent processes attributed to the host defence response to injuries. A synchronized activity of both pathways represents an essential prerequisite for restitution of host homeostasis after ultimate disturbances of the latter. Crosstalk between coagulation and inflammation is considered to inherit from primitive coagulation systems similar to invertebrates. For instance, in Horseshoe "crabs" (*Limulus*) coagulogen, a clotting protein with the bactericidal function, and coagulationrelated serine proteases are both located in circulating hemolymph cells (hemocytes) and are capable of simultaneously protecting against injury, as well as to isolate pathogens within cysts (Tanaka et al., 2009). In humans, the complement system as a part of innate immunity remains closely related to the hemostasis system (Markiewski et al., 2007). The organizations of these two systems demonstrate several similarities of both structural and functional features. Both systems are organized into proteolytic cascades; serine proteases of the chymotrypsin family are the components of the latter. Proteases of each system are, in their molecular structure, glycoproteins, which have a highly conservative catalytic site composed of serine, histidine, and aspartic amino acid residues. These proteases exist in the form of inactive zymogens and are subsequently activated by upstream, active proteases. Proteins of the complement and hemostasis systems are mostly synthesized in the liver by hepatocytes, besides being additionally produced during an acute-phase response stimulated by common inflammatory mediators. The biological role of the complement system is mentioned here in order to note that the above-mentioned similarities represent a forcible argument in favour of the common origin of both immunity and hemostasis phenomena. The complement system has been described in detail recently (Castellheim et al., 2009; Markiewski et al., 2007) and will not be further discussed here. Though, it is worthwhile to mention that the blood hemostasis system in humans, hypothetically, has been evolved progressively from the archaic innate immunity organization, diverging to certain narrow-specific pathways, which are responsible for the coagulation control.

Hemostasis in humans is organized as closely interrelated enzyme cascade systems: (i) fibrin clotting system (coagulation); (ii) multilevel system for preventing uncontrolled fibrin formation (anticoagulation); and (iii) system for limiting the amount of cross-linked fibrin (fibrinolysis). Together, coagulation, anticoagulation and fibrinolysis are associated into a

Hemostatic Soluble Plasma Proteins During Acute-Phase Response and Chronic Inflammation 107

The capacity of inflammatory mediators to regulate hemostasis, as well as some aspects of the coagulation ability to affect inflammatory events have been extensively reviewed (Butenas et al., 2009; Danese et al., 2010; Esmon, 2005; Jennewein et al., 2011; Levi et al., 2004; Medcalf, 2007). Reciprocal regulation of gene expression is the most important mechanism, by which inflammatory and hemostatic pathways interact with each other. The role of cell surface receptors in providing APP-associated signalling has been recently elucidated (Busso et al., 2008; Guitton et al., 2011). The role of the increased HSPPs in the regulation of hemostasis and inflammation pathways under pathophysiological conditions, however, remains less clear. The molecular mechanisms, responsible for the influence of inflammation upon hemostasis and *vice versa*, remain largely unknown. We would like to review here the most important post-translational events, which might perturb HSPPs structure and functions, as well as those influencing measurable levels of HSPPs during inflammation.

**2. Cooperation of inflammatory and hemostatic pathways during acute phase**

In normal conditions, HSPPs are presented by a soluble fibrous protein, fibrinogen, abundant serine protease zymogens (inactive enzymatic precursors), and minute amounts of active proteases, as well as by non-enzymatic cofactors and protease inhibitors (Table 1).

substrate for

of a fibrin clot

activation, not enzymatically active

as a cofactor for Xa participates in thrombin

cofactor for IXa in conversion of X to Xa, receptor for IXa and X, not enzymatically active

repair

to Xa

The basic function in hemostasis

polymerization to fibrin that is important in tissue

the conversion of Fg to Fn leading to the formation

the catalytic component of the extrinsic tenase, activates IX to IXa and X

the enzyme component of the intrinsic tenase, activates X to Xa

the enzyme component of the prothrombinase is

Proteolytic activation

by prothrombinase

by thrombin,

by XIa or TF-VIIa/PL-

by IXa-VIIIa/PL-

by thrombin increase,

by thrombin ND

IXa, Xa, XIIa ND

by thrombin increase

Ca2+ ND

Ca2+, ND

Expression during the acute-phase response

1,5-4,0-fold

no change or weak increase

**response**

**Clotting factors**  Fibrinogen (Fg) → fibrin (Fn)

Prothrombin (II) → thrombin (IIa)

Factor V (V) → activated factor V (Va)

Factor VII (VII) → activated factor VIIa

Factor VIII (VIII) → activated factor VIIIa

Factor IX (IX) → activated factor IXa

Factor X (X) → activated factor Xa

(VIIa)

(VIIIa)

(IXa)

Precursor conversion HSPP

identification

a fibrous (structural) protein

trypsin-like serine protease (endopeptidase)

ceruloplasminlike binding protein

serine protease (endopeptidase)

ceruloplasminlike binding protein

serine protease (endopeptidase)

serine protease (endopeptidase)

self-regulated and highly organized molecular machine that provides either acceleration or reduction of blood hemostasis process (Spronk et al., 2003). Accurate regulation of the coagulation rate is an effective mechanism, preventing circulatory disorders. This regulatory mechanism is known to be a part of the acute phase of the inflammatory reaction which provides rapid restoration of physiological homeostasis.

Hemostasis is closely linked to innate immunity and inflammation through particular regulatory coupling like protein C system (Fig. 1). Cooperation between hemostasis and innate immunity facilitates injury recognition, vessel wall reparation, and prevention of excessive bleeding without causing thrombosis. These processes are mediated by a balance of cellular surface components, cell-derived factors, and soluble plasma proteins (SPPs). After interruption of the vascular integrity, concentrations of some hemostatic SPPs (HSPPs) change in a manner typical of those of acute-phase proteins (APPs). In agreement with definition of APPs (Kushner & Rzewnicki, 1994; Morley & Kushner, 1982), at least several HSPPs including fibrinogen, plasminogen, and PAI-1 should also be classified as APPs, since: (i) the intensity of synthesis of these HSPPs dramatically changes (by more than 50%) during pathological processes (ii) HSPPs synthesis in hepatocytes during the acute-phase response are regulated by inflammatory-associated cytokines; (iii) due to chronic stimulation by inflammatory mediators, HSPPs may persist in circulation and participate in the formation of a "semantically paradoxical chronic acute-phase response" (Gabay & Kushner, 2001). The above-indicated similarities between inflammation and hemostasis determine their unidirectional changes. These changes involve pro-coagulant activities of inflammatory processes, as well as the pro-inflammatory efficacy of the hemostatic molecular machine.

Fig. 1. Schematic representation of the protein C-dependent cross-point between hemostatic and inflammatory pathways. Diagram illustrates a putative regulatory mechanism, maintaining homeostasis within physiological limits. Both pathways are balanced by the PC/APC (protein C/activated protein C) system, directly by attenuation of production of thrombin as a pro-inflammatory and pro-coagulant agent, or indirectly, by controlling NFkB-dependent anti-inflammatory pathways through EPCRs (endothelial protein C receptors) and PARs (protease-activated receptors) at the surface of the target cells. See the text for discussion in detail.

self-regulated and highly organized molecular machine that provides either acceleration or reduction of blood hemostasis process (Spronk et al., 2003). Accurate regulation of the coagulation rate is an effective mechanism, preventing circulatory disorders. This regulatory mechanism is known to be a part of the acute phase of the inflammatory reaction which

Hemostasis is closely linked to innate immunity and inflammation through particular regulatory coupling like protein C system (Fig. 1). Cooperation between hemostasis and innate immunity facilitates injury recognition, vessel wall reparation, and prevention of excessive bleeding without causing thrombosis. These processes are mediated by a balance of cellular surface components, cell-derived factors, and soluble plasma proteins (SPPs). After interruption of the vascular integrity, concentrations of some hemostatic SPPs (HSPPs) change in a manner typical of those of acute-phase proteins (APPs). In agreement with definition of APPs (Kushner & Rzewnicki, 1994; Morley & Kushner, 1982), at least several HSPPs including fibrinogen, plasminogen, and PAI-1 should also be classified as APPs, since: (i) the intensity of synthesis of these HSPPs dramatically changes (by more than 50%) during pathological processes (ii) HSPPs synthesis in hepatocytes during the acute-phase response are regulated by inflammatory-associated cytokines; (iii) due to chronic stimulation by inflammatory mediators, HSPPs may persist in circulation and participate in the formation of a "semantically paradoxical chronic acute-phase response" (Gabay & Kushner, 2001). The above-indicated similarities between inflammation and hemostasis determine their unidirectional changes. These changes involve pro-coagulant activities of inflammatory processes, as well as the pro-inflammatory efficacy of the hemostatic

Fig. 1. Schematic representation of the protein C-dependent cross-point between hemostatic

receptors) and PARs (protease-activated receptors) at the surface of the target cells. See the

and inflammatory pathways. Diagram illustrates a putative regulatory mechanism, maintaining homeostasis within physiological limits. Both pathways are balanced by the PC/APC (protein C/activated protein C) system, directly by attenuation of production of thrombin as a pro-inflammatory and pro-coagulant agent, or indirectly, by controlling NF-

kB-dependent anti-inflammatory pathways through EPCRs (endothelial protein C

provides rapid restoration of physiological homeostasis.

molecular machine.

text for discussion in detail.

The capacity of inflammatory mediators to regulate hemostasis, as well as some aspects of the coagulation ability to affect inflammatory events have been extensively reviewed (Butenas et al., 2009; Danese et al., 2010; Esmon, 2005; Jennewein et al., 2011; Levi et al., 2004; Medcalf, 2007). Reciprocal regulation of gene expression is the most important mechanism, by which inflammatory and hemostatic pathways interact with each other. The role of cell surface receptors in providing APP-associated signalling has been recently elucidated (Busso et al., 2008; Guitton et al., 2011). The role of the increased HSPPs in the regulation of hemostasis and inflammation pathways under pathophysiological conditions, however, remains less clear. The molecular mechanisms, responsible for the influence of inflammation upon hemostasis and *vice versa*, remain largely unknown. We would like to review here the most important post-translational events, which might perturb HSPPs structure and functions, as well as those influencing measurable levels of HSPPs during inflammation.

#### **2. Cooperation of inflammatory and hemostatic pathways during acute phase response**

In normal conditions, HSPPs are presented by a soluble fibrous protein, fibrinogen, abundant serine protease zymogens (inactive enzymatic precursors), and minute amounts of active proteases, as well as by non-enzymatic cofactors and protease inhibitors (Table 1).

Hemostatic Soluble Plasma Proteins During Acute-Phase Response and Chronic Inflammation 109

inhibits IIa, APC Pm, kallikrein, a remover of plasma enzymes

covalent complexes interfered with the binding of Pg(Pm) to Fn

inhibitor of fibrinolysis, removes Pg-binding sites

fibrinolysis by attenuation of Pm production

serpin inhibitor of Pm, forms

on Fn

serpin the major inhibitor of tPA

serpin inhibitor of urokinase as

serpin the major inhibitor of APC

Table 1. The main characteristics of hemostatic soluble plasma proteins (HSPPs)

anticoagulation; thus, these factors are called Janus-faced proteins.

that regulates the

as well as tPA and

Physiological anticoagulants are also available to suppress appropriate clotting factors. Some of the clotting factors (like thrombin or factor V) can promote both coagulation and

A hemostatic response to the activating signal is manifested by a series of transformations of proenzymes to activated enzymes in a cascade-like manner. The formation of thrombin at the final stage of the coagulation cascade is aimed at conversion of soluble fibrinogen into insoluble fibrin, the non-cellular matrix of blood thrombus. The thrombus formation is considerably accelerated due to accumulation of tissue factor (TF) at the sites of vascular endothelial damage. Tissue factor, a membrane-bound glycoprotein, is considered the common physiologic trigger of both hemostasis and inflammation pathways. Under normal conditions, none cells, which contact with the bloodstream, express TF.  Immediately upon exposure to the bloodstream, TF contacts with activated coagulation factor VIIa (VIIa), whose trace amounts (about 1 % of total inactive enzyme precursor, coagulation factor VII) are conventionally present in circulation. The formation of macromolecular complex TF-VIIa is a crucial event that initiates the first stage of the coagulation process, initiation phase. Alongside with that TF initiates local inflammatory

The basic function in hemostasis Proteolytic activation

by thrombin, plasmin,

well as tPA - ND

urokinase - ND

Expression during the acute-phase response

about 100-fold

trypsin ND

Precursor conversion HSPP

α2-macroglobulin

α2-antiplasmin (α2 – plasmin inhibitor)

Thrombin activable fibrinolytic inhibitor (TAFI) → activated form (TAFIa)

Plasminogen activator inhibitor of type 1

Plasminogen activator inhibitor of type2

Plasminogen activator inhibitor of type3 (PAI-3 or PCI)

(α2-M)

(α2 -PI)

(PAI-1)

(PAI-2)

reaction.

identification

inhibitor (α l -PI) inhibits IIa and APC

broad-range protease inhibitor

carboxypeptidase

thrombin activation

pathway

cascade

V, vWF

serpin Inhibition of VIIa, IXa, Xa

Protein S (PS) binding protein as cofactor for APC - increase

The basic function in hemostasis

the conversion of IX to IXa within the intrinsic

initiation of the intrinsic coagulation pathway *via* conversion XI to XIa

solution phase activation of kallikrein, factor VII and the complement

stabilization of the fibrin clot *via* cross linking the α and γ-chains of Fn, α2-PI,

reverse inhibition of Xa and IIa, then TF-VIIa independently from Ca2+

and XIa, kallikrein,

inactivation of Va and VIIIa, that inhibits the prothrombinase and tenase and, finally, IIa

the main endotheliumderived activator of the fibrinolytic system, converts Pg to Pm

responsible for the fibrin

activator of the Pg

serpin protects tissues from

clot digestion by t-PA, u-PA,

Proteolytic activation

TF-VIIa/PL-Ca2+

by surface

bound α-XIIa ND

by kallikrein decrease

by kallikrein decrease

by thrombin ND

plasmin, IIa - decrease

by α-thrombin/thrombomodulin, by Xa or IIa without Ca2+

by plasmin

elastase, XIIa

conversion to Pm - increase

proteolytic enzymes, - increase

no change or decrease

decrease or weak increase

> increase, 2-3- fold

Expression during the acute-phase response

Precursor conversion HSPP

Factor XI (X) → activated factor XIa(XIa)

Factor XII (XII) → activated factor XII

Factor α-XIIa (α-XIIa) → factor β-XIIa (β-XIIa)

Factor XIII (XIII) → activated factor XIIIa

**Anticoagulants**  Tissue factor pathway inhibitor (TFPI)

Antithrombin

Protein C (PC)→ Activated protein C

**Fibrinolytic proteins**

plasminogen activator, single chain form (sctPA) → active twochain form (tc-tPA)

Glu-plasminogen (Glu-Pg) → plasmin (Pm)

Urokinase-type plasminogen activator, single chain (sc-uPA)

**Proteinase inhibitors**  α1-Antitrypsin or alpha 1-proteinase

Tissue-type

(AT)

(APC)

(α-XIIa)

(XIIIa)

identification

(Xa) responsible for rapid

serine protease (endopeptidase)

serine protease (endopeptidase)

serine protease (endopeptidase)

transglutaminase

Kunitz-type protease inhibitor

trypsin-like serine protease (endopeptidase)

serine protease (endopeptidase)

serine protease (endopeptidase)

serine protease (endopeptidase)

(transpeptidase)

Table 1. The main characteristics of hemostatic soluble plasma proteins (HSPPs)

Physiological anticoagulants are also available to suppress appropriate clotting factors. Some of the clotting factors (like thrombin or factor V) can promote both coagulation and anticoagulation; thus, these factors are called Janus-faced proteins.

A hemostatic response to the activating signal is manifested by a series of transformations of proenzymes to activated enzymes in a cascade-like manner. The formation of thrombin at the final stage of the coagulation cascade is aimed at conversion of soluble fibrinogen into insoluble fibrin, the non-cellular matrix of blood thrombus. The thrombus formation is considerably accelerated due to accumulation of tissue factor (TF) at the sites of vascular endothelial damage. Tissue factor, a membrane-bound glycoprotein, is considered the common physiologic trigger of both hemostasis and inflammation pathways. Under normal conditions, none cells, which contact with the bloodstream, express TF.  Immediately upon exposure to the bloodstream, TF contacts with activated coagulation factor VIIa (VIIa), whose trace amounts (about 1 % of total inactive enzyme precursor, coagulation factor VII) are conventionally present in circulation. The formation of macromolecular complex TF-VIIa is a crucial event that initiates the first stage of the coagulation process, initiation phase. Alongside with that TF initiates local inflammatory reaction.

Hemostatic Soluble Plasma Proteins During Acute-Phase Response and Chronic Inflammation 111

Fig. 2. Schematic representation of the regulatory crosstalk between hemostasis and

circulating plasma whereas TF is the only cell surface glycoprotein shown here). The interconnection of coagulation and inflammation pathways is an essential prerequisite for host homeostasis restitution after injury. Coagulation factors (as shown here with pink circles) and inflammatory mediators (blue circles) promote both coagulation and inflammation through complex and reciprocal interactions, thereby sustaining the acute phase response. At least, two clotting factors, factor VIIa and thrombin, contribute in proinflammatory action of coagulation system through positive feed-back autoactivation mechanism (closed-loop arrows). Red curved arrow represents propagation phase of coagulation, blue curved arrow represents amplification of inflammation. In proportion to increasing levels of clotting and inflammatory APP, anticoagulation and anti-inflammation processes are activated. The limiting action of the anticoagulant and fibrinolytic systems on coagulation as well as anti-inflammatory mechanism of inflammation attenuation is depicted by brick-built barrier. Abbreviations: IIa, VIIa, IXa, Xa and XIa indicate activated coagulation factors; Fg, fibrinogen; Fn, fibrin; TF, tissue factor; PARs, protease-activated receptors; IL, interleukin; CRP, C-reactive protein; ICAM, intercellular adhesion molecule-1; PC/APC, protein C/activated protein C; AT, antithrombin; TFPI, tissue factor pathway inhibitor; IL-1Ra, interleukin-1 receptor antagonist; TGF-ß, transforming growth factor beta.

inflammation in response to exposed TF (coagulation factors and cytokines are all present in

#### **2.1 Initiation phase of the coagulation process**

Tissue factor-VIIa complexes, newly appeared on the boundary between blood flow and the vessel wall, begin to bind plasma-derived coagulation factor VII to produce additionally factor VIIa. Thus, TF acts as a cofactor in the factor VII/VIIa autoactivation process (Fig. 2). Membrane-bound TF/VIIa complexes also interact with small amounts of coagulation factor X (X) and coagulation factor IX (IX). Activated factors X (Xa) and IX (IXa) start up prothrombin (coagulation factor II) conversion to thrombin (IIa). This first cycle of restricted thrombin production is limited by two plasma-derived inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT). The former one neutralizes factor Xa when forms a quaternary structure with TF-VIIa-Xa. As well, AT upon binding to heparan sulphate/heparin, rapidly inactivates free factors Xa, IXa, and thrombin, initially accumulated at the site of vascular injury. So, TF-VIIa itself is incapable to generate substantial amounts of thrombin during the initiation phase. However, the TF-dependent cycle of thrombin production can overcome inhibition by TFPI and AT, when TF is maintained at a sufficiently high level (Tanaka et al., 2009). Blood-borne forms of TF (soluble sTF or TF-positive microparticles) shed from disrupted cell membranes of different origin presumably can be an additional driver of the increased TF-initiated thrombin production (Sommeijer et al., 2006). Apparently, cell-exposed and blood-borne TF can promote transduction of inflammatory signals *via* cellular protease-activated receptors (PARs). For example, sTF-mediated inflammation in animal models might develop *via* platelet PAR-4 signalling, while TF-proteases complexes (TF-VIIa and TF-VIIa-Xa ) induce the activity of signalling pathways in vascular cells *via* PAR-1 and PAR-2 (Busso et al., 2008; Rao & Pendurthi, 2005; Riewald & Ruf, 2001). Being a mediator of both inflammatory and hemostatic pathways, TF integrates different extra cellular signals and cellular responses, thus participating in the development of a host acute-phase response (Fig. 2). As an extremely potent triggering molecule, TF is capable of translating injury signals into activation of the coagulation cascade, sustaining thrombin initiation, and promoting its propagation.

#### **2.2 Propagation phase of the coagulation process**

Trace amounts of thrombin promote formation of a IXa-VIIIa-Ca2+-phospholipid assembly (tenase complex) or a Xa-Va-Ca2+-phospholipid assembly (prothrombinase complex) *via* feedback activation of non-enzymatic cofactors VIII (VIIIa) and V (Va) after their binding to negatively charged phospholipids (phosphatidylinositol and phosphatidylserine) on the surface of activated platelets in the presence of calcium ions. Thrombin also activates factor XI (XIa), which additionally stimulates the tenase complex. Tenase-produced prothrombinase complexes lead to the explosive generation of thrombin, which ultimately leads to generation of a fibrin clot. During an episode of TF-initiated coagulation, tenase and prothrombinase complexes are generated in concentrations that might be sufficient to maintain the TF-independent procoagulant response as long, as the reactants are available. From this moment, normal coagulation may become fully independent of TF (Butenas et al., 2009). The propagation phase can continue and prolong the acute-phase response, where driving of thrombin generation is a requisite for an adequate bleeding prevention *via* more fibrin deposition. By binding to PARs, thrombin activates platelets, endothelial cells, and immune cells. As a result, cytokines and chemokines are additionally expressed, as well as certain HSPP secretion is intensified, leukocyte and platelet recruitment to inflammatory *foci* increases, and fibrin deposition is accumulated. These events considerably enhance local

Tissue factor-VIIa complexes, newly appeared on the boundary between blood flow and the vessel wall, begin to bind plasma-derived coagulation factor VII to produce additionally factor VIIa. Thus, TF acts as a cofactor in the factor VII/VIIa autoactivation process (Fig. 2). Membrane-bound TF/VIIa complexes also interact with small amounts of coagulation factor X (X) and coagulation factor IX (IX). Activated factors X (Xa) and IX (IXa) start up prothrombin (coagulation factor II) conversion to thrombin (IIa). This first cycle of restricted thrombin production is limited by two plasma-derived inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT). The former one neutralizes factor Xa when forms a quaternary structure with TF-VIIa-Xa. As well, AT upon binding to heparan sulphate/heparin, rapidly inactivates free factors Xa, IXa, and thrombin, initially accumulated at the site of vascular injury. So, TF-VIIa itself is incapable to generate substantial amounts of thrombin during the initiation phase. However, the TF-dependent cycle of thrombin production can overcome inhibition by TFPI and AT, when TF is maintained at a sufficiently high level (Tanaka et al., 2009). Blood-borne forms of TF (soluble sTF or TF-positive microparticles) shed from disrupted cell membranes of different origin presumably can be an additional driver of the increased TF-initiated thrombin production (Sommeijer et al., 2006). Apparently, cell-exposed and blood-borne TF can promote transduction of inflammatory signals *via* cellular protease-activated receptors (PARs). For example, sTF-mediated inflammation in animal models might develop *via* platelet PAR-4 signalling, while TF-proteases complexes (TF-VIIa and TF-VIIa-Xa ) induce the activity of signalling pathways in vascular cells *via* PAR-1 and PAR-2 (Busso et al., 2008; Rao & Pendurthi, 2005; Riewald & Ruf, 2001). Being a mediator of both inflammatory and hemostatic pathways, TF integrates different extra cellular signals and cellular responses, thus participating in the development of a host acute-phase response (Fig. 2). As an extremely potent triggering molecule, TF is capable of translating injury signals into activation of the coagulation cascade, sustaining thrombin initiation, and promoting its

Trace amounts of thrombin promote formation of a IXa-VIIIa-Ca2+-phospholipid assembly (tenase complex) or a Xa-Va-Ca2+-phospholipid assembly (prothrombinase complex) *via* feedback activation of non-enzymatic cofactors VIII (VIIIa) and V (Va) after their binding to negatively charged phospholipids (phosphatidylinositol and phosphatidylserine) on the surface of activated platelets in the presence of calcium ions. Thrombin also activates factor XI (XIa), which additionally stimulates the tenase complex. Tenase-produced prothrombinase complexes lead to the explosive generation of thrombin, which ultimately leads to generation of a fibrin clot. During an episode of TF-initiated coagulation, tenase and prothrombinase complexes are generated in concentrations that might be sufficient to maintain the TF-independent procoagulant response as long, as the reactants are available. From this moment, normal coagulation may become fully independent of TF (Butenas et al., 2009). The propagation phase can continue and prolong the acute-phase response, where driving of thrombin generation is a requisite for an adequate bleeding prevention *via* more fibrin deposition. By binding to PARs, thrombin activates platelets, endothelial cells, and immune cells. As a result, cytokines and chemokines are additionally expressed, as well as certain HSPP secretion is intensified, leukocyte and platelet recruitment to inflammatory *foci* increases, and fibrin deposition is accumulated. These events considerably enhance local

**2.1 Initiation phase of the coagulation process**

**2.2 Propagation phase of the coagulation process**

propagation.

Fig. 2. Schematic representation of the regulatory crosstalk between hemostasis and inflammation in response to exposed TF (coagulation factors and cytokines are all present in circulating plasma whereas TF is the only cell surface glycoprotein shown here). The interconnection of coagulation and inflammation pathways is an essential prerequisite for host homeostasis restitution after injury. Coagulation factors (as shown here with pink circles) and inflammatory mediators (blue circles) promote both coagulation and inflammation through complex and reciprocal interactions, thereby sustaining the acute phase response. At least, two clotting factors, factor VIIa and thrombin, contribute in proinflammatory action of coagulation system through positive feed-back autoactivation mechanism (closed-loop arrows). Red curved arrow represents propagation phase of coagulation, blue curved arrow represents amplification of inflammation. In proportion to increasing levels of clotting and inflammatory APP, anticoagulation and anti-inflammation processes are activated. The limiting action of the anticoagulant and fibrinolytic systems on coagulation as well as anti-inflammatory mechanism of inflammation attenuation is depicted by brick-built barrier. Abbreviations: IIa, VIIa, IXa, Xa and XIa indicate activated coagulation factors; Fg, fibrinogen; Fn, fibrin; TF, tissue factor; PARs, protease-activated receptors; IL, interleukin; CRP, C-reactive protein; ICAM, intercellular adhesion molecule-1; PC/APC, protein C/activated protein C; AT, antithrombin; TFPI, tissue factor pathway inhibitor; IL-1Ra, interleukin-1 receptor antagonist; TGF-ß, transforming growth factor beta.

Hemostatic Soluble Plasma Proteins During Acute-Phase Response and Chronic Inflammation 113

inactivates tPA, (ii) α2-antiplasmin, primary Pm inhibitor that prevents Pm-dependent nonspecific proteolysis due to effective neutralization of Pm, and (iii) thrombin activable fibrinolytic inhibitor (TAFI) that down-regulates the cofactor activity of fibrin during

The activities of both coagulation and fibrinolytic cascades are normally latent but have the potential to be accelerated in an extremely acute manner during inflammation. The coagulation and fibrinolysis pathways have to follow each other, retaining a delicate physiological balance preventing thrombosis and bleeding. Activation of the coagulation cascade leads to increases in the plasma levels of coagulation factors VIIa, Xa, and thrombin, which are pro-inflammatory factors contributing to the acute-phase response (Fig. 2). In addition, fibrin deposition and fibrin degradation products, FDPs, enhance inflammation. Coagulation factors elicit inflammation *via* affecting a number of blood/vascular cells through protease-activated receptor (PAR)-mediated pathways up-regulating the expression of numerous APPs (tumour necrosis factor-alpha, interleukins, adhesion molecules, and growth factors) (Chua, 2005). At least fibrin, thrombin, and coagulation factor Xa, all are important cell-signalling effector molecules that are responsible for receptor triggering. When PARs are activated constantly (e.g., under the action of repeated stimuli), the acutephase response can be inverted into a chronic one. Therefore, the inflammatory consequence caused by coagulation should be abolished within a necessary time intervals; otherwise it could be enormous. Resolution of the acute-phase response requires down-regulation of inflammatory/procoagulant APPs expression. In particular, IL-4, IL-10, TGF- ß are antiinflammatory agents that inhibit the production of numerous inflammatory cytokines, including TNF-α, IL-1ß, IL-6, IL-8, and, finally, IL-10 itself (de Waal et al., 1991; Walley et al., 1996). In fact, human blood monocytes are known to produce both pro- and antiinflammatory cytokines. During resolution, monocytes and macrophages considerably increase production of the latter above the former, thereby preventing prolongation or escalation of an early inflammatory response (Fig. 2). The concentration of cytokine-induced

procoagulants is reciprocally decreased; thus, vascular homeostasis is restored.

*versa* — hypercoagulation follows amplification of inflammation (Levi et al., 2004)

inflammatory bowel disease, and lung and heart inflammation (Fig. 3).

A failure of the control of these processes causes incorrect inflammation termination or even its propagation. Such an inconsistency leads to deregulation of hemostasis, which, in turn, might force the further leap of inflammatory responses. Under pathological conditions, cytokines are released by immune regulatory cells in sites of the local inflammatory response. This process may be acute but limited in time (reverting to the normal homeostatic state) or persistent (resulting in chronic activation of coagulation and fibrinolysis). Initially acting within the frame of the adaptive defence system, inflammation and hemostasis might develop from a local response to a systemic host reaction. Escalation of inflammation can induce endothelial dysfunction subsequently activating the coagulation cascade, and *vice*

Under these conditions mutual activation of coagulation and fibrinolysis might follow to potentially exhausting consumptive coagulopathy and disseminated intravascular coagulation. A detrimental inflammatory response resulting from coupling of procoagulant and pro-inflammatory stimuli might cause thrombophilia, and, furthermore, provoke the thrombotic events. In such a way, inflammation/coagulation interaction drastically increases a risk of thrombus formation implicated in the pathogenesis of several diseases in humans. On the one hand, these are thrombophilias, atherosclerosis, and other cardiovascular pathologies, as well as intercurrent illnesses (like trauma and cancer) or surgery. On the other hand, these are acute/chronic inflammatory diseases, including sepsis,

activation of plasminogen and, thereby, suppresses fibrinolysis.

inflammation and coagulation. Furthermore, the formation of thrombi in the microvasculature provides a mechanical barrier that blocks spreading of inflammatory/coagulation mediators into the circulation. Such limited clotting restricts thrombus propagation and prevents from acute local inflammation turning into systemic complications. An anti-clotting molecular process induced by several physiological anticoagulants and fibrinolytic agents is designated for regulation of an adequate clot size and formation of an effective thrombus. Two hemostatic pathways, anticoagulation and fibrinolysis, both are responsible for coagulation termination (Fig. 2).

#### **2.3 Termination phase of the coagulation process**

After bleeding is arrested, and the injured vessel is repaired, coagulation attenuation begins to dominate over its propagation due to accumulation of inhibitors of blood clotting. Natural clotting inhibitors are orchestrated through successive induction of three major anticoagulantdependent pathways: tissue factor pathway inhibitor-, heparin/antithrombin- and the protein C-dependent pathways. A tissue factor pathway inhibitor (TFPI), as was mentioned above, is a most effective inhibitor of coagulation at the initiation phase. This inhibitor specifically blocks the TF-VIIa-Xa complex after trace factor Xa has been formed (Spronk et al., 2003). Antithrombin (AT) is a direct protease inhibitor, which attenuates accumulation of coagulation factors IXa, Xa, and IIa during the propagation phase. Heparin-like glycosaminoglycans accelerate the rate of inactivation of these clotting factors by AT. The protein C system provides multi-directional attenuation of thrombin procoagulant activity and this terminates coagulation. Thrombomodulin (TM), a endothelial cell membrane-associated protein, is capable of to tackling excessive thrombin, thus changing its specific enzyme activity. Within the thrombin-thrombomodulin complex, thrombin looses its affinity to fibrinogen or cellular PARs. Instead this, it possesses an ability to convert precursor protein C (PC) into an anticoagulant enzyme, activated protein C (APC). Activated protein C interrupts thrombin propagation *via* limited proteolysis of cofactors Va and VIIIa. Cofactor protein S and platelet membrane phospholipids provide manifold acceleration of the APC activity. Endothelial protein C receptors (EPCR) enhance the thrombin-thrombomodulin affinity to PC. In such a way the protein C system down-regulates the coagulation cascade to moderate the explosive trend of thrombin production. Its anticoagulant competence enhances, due to the modulation of the thrombin activity through two mechanisms, inhibition of prothrombin converting into thrombin and promotion of thrombin inversion from a procoagulant enzyme into an anticoagulant one. Inhibition of thrombin formation can also reduce thrombin's proinflammatory activities (Sarangi et al., 2010).

In a complementary mode with respect to anticoagulation, the surplus clots are dissolved by proteases of the fibrinolytic system. Activation of intravascular fibrinolysis is controlled through enhancing synthesis and secretion of tissue plasminogen activator (tPA) by endothelial cells during fibrin clotting. Tissue plasminogen activator is released into the clot and binds in the clot volume with fibrin(ogen) and plasminogen (Pg). The formation of ternary Fn-tPA-Pg complex extremely effectively accelerates Pg converting into the serine protease plasmin (Pm). Plasmin cleaves fibrin into soluble fragments, the so called fibrin degradation products (FDPs). The rate and extent of local delivery of tPA during the clot formation is important for enhancing the process of fibrinolysis (Schrauwen et al., 1994). To avoid excessive clot digestion, which can affect bleeding, the activity of fibrinolytic system is down-regulated by numerous plasma- and cell-derived inhibitors (Meltzer et al., 2010). These are (i) plasminogen activator inhibitor of type 1 (PAI-1) that highly specifically

inflammation and coagulation. Furthermore, the formation of thrombi in the microvasculature provides a mechanical barrier that blocks spreading of inflammatory/coagulation mediators into the circulation. Such limited clotting restricts thrombus propagation and prevents from acute local inflammation turning into systemic complications. An anti-clotting molecular process induced by several physiological anticoagulants and fibrinolytic agents is designated for regulation of an adequate clot size and formation of an effective thrombus. Two hemostatic pathways, anticoagulation and

After bleeding is arrested, and the injured vessel is repaired, coagulation attenuation begins to dominate over its propagation due to accumulation of inhibitors of blood clotting. Natural clotting inhibitors are orchestrated through successive induction of three major anticoagulantdependent pathways: tissue factor pathway inhibitor-, heparin/antithrombin- and the protein C-dependent pathways. A tissue factor pathway inhibitor (TFPI), as was mentioned above, is a most effective inhibitor of coagulation at the initiation phase. This inhibitor specifically blocks the TF-VIIa-Xa complex after trace factor Xa has been formed (Spronk et al., 2003). Antithrombin (AT) is a direct protease inhibitor, which attenuates accumulation of coagulation factors IXa, Xa, and IIa during the propagation phase. Heparin-like glycosaminoglycans accelerate the rate of inactivation of these clotting factors by AT. The protein C system provides multi-directional attenuation of thrombin procoagulant activity and this terminates coagulation. Thrombomodulin (TM), a endothelial cell membrane-associated protein, is capable of to tackling excessive thrombin, thus changing its specific enzyme activity. Within the thrombin-thrombomodulin complex, thrombin looses its affinity to fibrinogen or cellular PARs. Instead this, it possesses an ability to convert precursor protein C (PC) into an anticoagulant enzyme, activated protein C (APC). Activated protein C interrupts thrombin propagation *via* limited proteolysis of cofactors Va and VIIIa. Cofactor protein S and platelet membrane phospholipids provide manifold acceleration of the APC activity. Endothelial protein C receptors (EPCR) enhance the thrombin-thrombomodulin affinity to PC. In such a way the protein C system down-regulates the coagulation cascade to moderate the explosive trend of thrombin production. Its anticoagulant competence enhances, due to the modulation of the thrombin activity through two mechanisms, inhibition of prothrombin converting into thrombin and promotion of thrombin inversion from a procoagulant enzyme into an anticoagulant one. Inhibition of thrombin formation can also reduce thrombin's pro-

In a complementary mode with respect to anticoagulation, the surplus clots are dissolved by proteases of the fibrinolytic system. Activation of intravascular fibrinolysis is controlled through enhancing synthesis and secretion of tissue plasminogen activator (tPA) by endothelial cells during fibrin clotting. Tissue plasminogen activator is released into the clot and binds in the clot volume with fibrin(ogen) and plasminogen (Pg). The formation of ternary Fn-tPA-Pg complex extremely effectively accelerates Pg converting into the serine protease plasmin (Pm). Plasmin cleaves fibrin into soluble fragments, the so called fibrin degradation products (FDPs). The rate and extent of local delivery of tPA during the clot formation is important for enhancing the process of fibrinolysis (Schrauwen et al., 1994). To avoid excessive clot digestion, which can affect bleeding, the activity of fibrinolytic system is down-regulated by numerous plasma- and cell-derived inhibitors (Meltzer et al., 2010). These are (i) plasminogen activator inhibitor of type 1 (PAI-1) that highly specifically

fibrinolysis, both are responsible for coagulation termination (Fig. 2).

**2.3 Termination phase of the coagulation process**

inflammatory activities (Sarangi et al., 2010).

inactivates tPA, (ii) α2-antiplasmin, primary Pm inhibitor that prevents Pm-dependent nonspecific proteolysis due to effective neutralization of Pm, and (iii) thrombin activable fibrinolytic inhibitor (TAFI) that down-regulates the cofactor activity of fibrin during activation of plasminogen and, thereby, suppresses fibrinolysis.

The activities of both coagulation and fibrinolytic cascades are normally latent but have the potential to be accelerated in an extremely acute manner during inflammation. The coagulation and fibrinolysis pathways have to follow each other, retaining a delicate physiological balance preventing thrombosis and bleeding. Activation of the coagulation cascade leads to increases in the plasma levels of coagulation factors VIIa, Xa, and thrombin, which are pro-inflammatory factors contributing to the acute-phase response (Fig. 2). In addition, fibrin deposition and fibrin degradation products, FDPs, enhance inflammation. Coagulation factors elicit inflammation *via* affecting a number of blood/vascular cells through protease-activated receptor (PAR)-mediated pathways up-regulating the expression of numerous APPs (tumour necrosis factor-alpha, interleukins, adhesion molecules, and growth factors) (Chua, 2005). At least fibrin, thrombin, and coagulation factor Xa, all are important cell-signalling effector molecules that are responsible for receptor triggering. When PARs are activated constantly (e.g., under the action of repeated stimuli), the acutephase response can be inverted into a chronic one. Therefore, the inflammatory consequence caused by coagulation should be abolished within a necessary time intervals; otherwise it could be enormous. Resolution of the acute-phase response requires down-regulation of inflammatory/procoagulant APPs expression. In particular, IL-4, IL-10, TGF- ß are antiinflammatory agents that inhibit the production of numerous inflammatory cytokines, including TNF-α, IL-1ß, IL-6, IL-8, and, finally, IL-10 itself (de Waal et al., 1991; Walley et al., 1996). In fact, human blood monocytes are known to produce both pro- and antiinflammatory cytokines. During resolution, monocytes and macrophages considerably increase production of the latter above the former, thereby preventing prolongation or escalation of an early inflammatory response (Fig. 2). The concentration of cytokine-induced procoagulants is reciprocally decreased; thus, vascular homeostasis is restored.

A failure of the control of these processes causes incorrect inflammation termination or even its propagation. Such an inconsistency leads to deregulation of hemostasis, which, in turn, might force the further leap of inflammatory responses. Under pathological conditions, cytokines are released by immune regulatory cells in sites of the local inflammatory response. This process may be acute but limited in time (reverting to the normal homeostatic state) or persistent (resulting in chronic activation of coagulation and fibrinolysis). Initially acting within the frame of the adaptive defence system, inflammation and hemostasis might develop from a local response to a systemic host reaction. Escalation of inflammation can induce endothelial dysfunction subsequently activating the coagulation cascade, and *vice versa* — hypercoagulation follows amplification of inflammation (Levi et al., 2004)

Under these conditions mutual activation of coagulation and fibrinolysis might follow to potentially exhausting consumptive coagulopathy and disseminated intravascular coagulation. A detrimental inflammatory response resulting from coupling of procoagulant and pro-inflammatory stimuli might cause thrombophilia, and, furthermore, provoke the thrombotic events. In such a way, inflammation/coagulation interaction drastically increases a risk of thrombus formation implicated in the pathogenesis of several diseases in humans. On the one hand, these are thrombophilias, atherosclerosis, and other cardiovascular pathologies, as well as intercurrent illnesses (like trauma and cancer) or surgery. On the other hand, these are acute/chronic inflammatory diseases, including sepsis, inflammatory bowel disease, and lung and heart inflammation (Fig. 3).

Hemostatic Soluble Plasma Proteins During Acute-Phase Response and Chronic Inflammation 115

Recently, a clear association of high APP levels with a human procoagulant phenotype and impaired fibrinolysis were found in some studies. Indeed, a certain relationship is believed between the plasma levels of C-reactive protein (CRP) and some HSPPs. C-reactive protein is at present used as a sensitive biomarker of acute and chronic inflammation. This is the only APP that correctly displays the severity of vascular pathologies: from an endotheliumderived focal inflammatory response to a hard coronary lesion (Calabrò et al., 2009; Willerson & Ridker, 2004). CRP levels are now detected using a high-sensitivity assay (hsCRP); these indices are found to be an accurate predictor of cardiovascular disease (CVD)

The procoagulant function of the C-reactive protein is still debated (MacCallum, 2005), but there is some evidence proving that CRP is associated with the metabolism of HSPP. In *ex vivo* experiments on cell systems, CRP was found to induce expression of inflammatory cytokines or TF in monocytes (Cermak et al., 1993), of thrombomodulin and von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVECs) (Blann & Lip, 2003), and of PAI-1 in human arterial endothelial cells (HAECs) (Chen et al., 2008; Devaraj et al., 2003). Close correlation of the CRP amount with increasing fibrinogen levels was found in patients with acute ischemic stroke (Tamam et al., 2005). The CRP expression reflects not only to be a predictor but rather an active mediator of atherothrombotic events, as was reported for *in vivo* CRP-dependent induction of TF in blood monocytes (Sardo et al., 2008). Increased levels of hsCRP are associated with such CVD, as severe unstable angina, myocardial infarction, stroke, and peripheral arterial disease (de Ferranti & Rifai, 2007). The causative role of CRP in thrombogenesis is at present believed doubtful, but its active participation is supported by some results described earlier. One of the small group evaluation reports revealed that activation of coagulation and fibrinolysis induced by recombinant CRP infusion provoked increases in the levels of prothrombin F1+2 and D-dimer, as well as in the vWF and PAI-1 concentrations (Bisoendial et al., 2005). CRP also attenuated the fibrinolytic capacity, by inhibiting the tPA activity and stimulating PAI-1. An increased ECLT (euglobulin clot lysis time) and, hence, a decreased fibrinolytic capacity in the blood plasma obtained from volunteers with high CRP levels were found (Zouaoui Boudjeltia et al., 2004). These data confirm a conclusion on down-regulation of fibrinolysis during the enhanced inflammatory

The studies that elucidate the inhibitory role of cytokines in fibrinolysis are not limited to that of CRP. A multifunctional cytokine, IL-1, was shown to stimulate up-regulation of specific mRNA expression of urokinase-type plasminogen activator (u-PA) (Wojta et al., 1994). IL-1 also increased accumulation of PAI-1 in cardiac microvascular endothelial cells (Okada et al., 1998) and also controlled expression of PAI-1 and u-PA in human astrocytes (Kasza et al., 2002). Production of PAI-1 protein in human adult cardiac myocytes was found to be increased up to two times by interleukin-1α and tumour necrosis factor-α (TNF-α) and up to five times by transforming growth factor-ß (TGF- ß ) and oncostatin M. However, t-PA production in human cardiac myocytes did not change after cytokine treatment (Macfelda et al., 2002). By contrast, IL-1 and tissue necrosis factor alpha inhibited t-PA in HUVEC

During severe inflammation, the function of fibrinolysis can be impaired. The same is true with respect to anticoagulant pathways. It was recently documented that an increase in serum CRP level in dogs was accompanied by lowering of the AT concentration (Cheng et

**3. Pro-inflammatory environment sustains coagulation**

(de Ferranti & Rifai, 2007).

response indicated by CRP.

(Bevilacqua et al., 1986).

Fig. 3. A simplified hypothetic model of pathophysiological interactions between inflammatory and hemostatic APPs. Each spiral turn represents a potentially vicious cycle driven by excessive concentrations of some components (this is shown as arrows up) and/or insufficient concentrations of other components (as arrows down). Amplification of HSPP activation during an initiation phase is exerted through interaction with the components of the innate immune system, which, in turn, prolongs inflammation during the propagation phase. As a result, both processes, coagulation and inflammation, can come into perpetuation phase. These complex interactions can lead to life-threatening complications, such as thrombosis or sepsis. Refer to the text for discussion in detail.

Abbreviations: IIa, VIIa, IXa, Xa, and XIa indicate activated coagulation factors; Va and VIIIa – non enzymatic cofactors; Fn, fibrin; TF, tissue factor; sTF, blood-borne (soluble) forms of tissue factor; PARs, protease-activated receptors; IL, interleukin; CRP, C-reactive protein; TNF−α, tumour necrosis factor alpha; tPA, tissue activator of plasminogen; APC, activated protein C; AT, antithrombin; PAI-1, plasminogen activator inhibitor of type 1; TAFI, thrombin activable fibrinolytic inhibitor.

#### **3. Pro-inflammatory environment sustains coagulation**

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

Fig. 3. A simplified hypothetic model of pathophysiological interactions between

phase. As a result, both processes, coagulation and inflammation, can come into

plasminogen activator inhibitor of type 1; TAFI, thrombin activable fibrinolytic inhibitor.

such as thrombosis or sepsis. Refer to the text for discussion in detail.

inflammatory and hemostatic APPs. Each spiral turn represents a potentially vicious cycle driven by excessive concentrations of some components (this is shown as arrows up) and/or insufficient concentrations of other components (as arrows down). Amplification of HSPP activation during an initiation phase is exerted through interaction with the components of the innate immune system, which, in turn, prolongs inflammation during the propagation

perpetuation phase. These complex interactions can lead to life-threatening complications,

Abbreviations: IIa, VIIa, IXa, Xa, and XIa indicate activated coagulation factors; Va and VIIIa – non enzymatic cofactors; Fn, fibrin; TF, tissue factor; sTF, blood-borne (soluble) forms of tissue factor; PARs, protease-activated receptors; IL, interleukin; CRP, C-reactive protein; TNF−α, tumour necrosis factor alpha; tPA, tissue activator of plasminogen; APC, activated protein C; AT, antithrombin; PAI-1, Recently, a clear association of high APP levels with a human procoagulant phenotype and impaired fibrinolysis were found in some studies. Indeed, a certain relationship is believed between the plasma levels of C-reactive protein (CRP) and some HSPPs. C-reactive protein is at present used as a sensitive biomarker of acute and chronic inflammation. This is the only APP that correctly displays the severity of vascular pathologies: from an endotheliumderived focal inflammatory response to a hard coronary lesion (Calabrò et al., 2009; Willerson & Ridker, 2004). CRP levels are now detected using a high-sensitivity assay (hsCRP); these indices are found to be an accurate predictor of cardiovascular disease (CVD) (de Ferranti & Rifai, 2007).

The procoagulant function of the C-reactive protein is still debated (MacCallum, 2005), but there is some evidence proving that CRP is associated with the metabolism of HSPP. In *ex vivo* experiments on cell systems, CRP was found to induce expression of inflammatory cytokines or TF in monocytes (Cermak et al., 1993), of thrombomodulin and von Willebrand factor (vWF) in human umbilical vein endothelial cells (HUVECs) (Blann & Lip, 2003), and of PAI-1 in human arterial endothelial cells (HAECs) (Chen et al., 2008; Devaraj et al., 2003). Close correlation of the CRP amount with increasing fibrinogen levels was found in patients with acute ischemic stroke (Tamam et al., 2005). The CRP expression reflects not only to be a predictor but rather an active mediator of atherothrombotic events, as was reported for *in vivo* CRP-dependent induction of TF in blood monocytes (Sardo et al., 2008). Increased levels of hsCRP are associated with such CVD, as severe unstable angina, myocardial infarction, stroke, and peripheral arterial disease (de Ferranti & Rifai, 2007). The causative role of CRP in thrombogenesis is at present believed doubtful, but its active participation is supported by some results described earlier. One of the small group evaluation reports revealed that activation of coagulation and fibrinolysis induced by recombinant CRP infusion provoked increases in the levels of prothrombin F1+2 and D-dimer, as well as in the vWF and PAI-1 concentrations (Bisoendial et al., 2005). CRP also attenuated the fibrinolytic capacity, by inhibiting the tPA activity and stimulating PAI-1. An increased ECLT (euglobulin clot lysis time) and, hence, a decreased fibrinolytic capacity in the blood plasma obtained from volunteers with high CRP levels were found (Zouaoui Boudjeltia et al., 2004). These data confirm a conclusion on down-regulation of fibrinolysis during the enhanced inflammatory response indicated by CRP.

The studies that elucidate the inhibitory role of cytokines in fibrinolysis are not limited to that of CRP. A multifunctional cytokine, IL-1, was shown to stimulate up-regulation of specific mRNA expression of urokinase-type plasminogen activator (u-PA) (Wojta et al., 1994). IL-1 also increased accumulation of PAI-1 in cardiac microvascular endothelial cells (Okada et al., 1998) and also controlled expression of PAI-1 and u-PA in human astrocytes (Kasza et al., 2002). Production of PAI-1 protein in human adult cardiac myocytes was found to be increased up to two times by interleukin-1α and tumour necrosis factor-α (TNF-α) and up to five times by transforming growth factor-ß (TGF- ß ) and oncostatin M. However, t-PA production in human cardiac myocytes did not change after cytokine treatment (Macfelda et al., 2002). By contrast, IL-1 and tissue necrosis factor alpha inhibited t-PA in HUVEC (Bevilacqua et al., 1986).

During severe inflammation, the function of fibrinolysis can be impaired. The same is true with respect to anticoagulant pathways. It was recently documented that an increase in serum CRP level in dogs was accompanied by lowering of the AT concentration (Cheng et

Hemostatic Soluble Plasma Proteins During Acute-Phase Response and Chronic Inflammation 117