YKL-40 amino acid and cDNA sequence

The complete amino acid and cDNA sequence of human YKL-40 was published by Hakala et al. in 1993 (GenBank Accession number: M80927). Human YKL-40 contains a single polypeptide chain of 383 amino acids and has a calculated molecular mass of 40,476 Da (Hakala et al. 1993) and an isoelectric point of about 7.6 (Renkema et al. 1998). The sequence of YKL-40 from several other mammals is known: pig (Shackelton et al. 1995) (84% sequence identity), cow (83%), goat (Mohanty et al. 2003) (83%), sheep (83%), guinea pig (De Ceuninck et al. 1998), rat (80%), and mouse (Morrison et al. 1994) (73% sequence identity). Amino acid sequence analysis reveals that YKL-40 belongs to the glycosyl hydrolase family 18 (Henrissat et al. 1993). This family consists of enzymes and proteins, and includes chitinases from various species (mammalian, bacteria, fungi, nematodes, insects and plants) (Aronson et al. 1997). Human YKL-40 shares significant amino acid sequence identity to bacterial chitinases (Hakala et al. 1993; Johansen et al. 1993 I) (31% sequence identity) and to seven other "mammalian chitinase-like proteins": 1) human oviduct-specific glycoprotein (OGP) (Arias et al. 1994; Buhi 2002) (46% sequence identity); 2) human chitotriosidase (Boot et al. 1995) (52%); 3) human YKL-39 (Hu et al. 1996) (51%); 4) human TSA 1902 (Saito et al. 1999) also named acidic mammalian chitinase (AMCase) (Boot et al. 2001) (51%); 5) mouse YM1 (Jin et al. 1998) (46%) also named eosinophil chemotactic cytokine (ECF-L) (Owhashi et al. 2000); 6) mouse chitinase like protein 2 (45%) (Ward et al. 2001) and 7) mouse protein MGC58999 (43% sequence identity). Three of these proteins have only been described in mouse. All 8 "mammalian chitinase-like proteins" show a high level of sequence identity over certain regions and strict conservation of several structurally important residues including proline and cysteine. The N-terminal amino acid sequence and the catalytic center are highly conserved (> 70% identical), whereas the identities are low in the C-terminal sequence. Interestingly, it has also been demonstrated that Drosophila melanogaster secretes several proteins, DS47 and imaginal disc growth factors (IDGFs), with sequence identity to YKL-40 (DS47: 34%; IDGFs: 16-23%) (Kirkpatrick et al. 1995; Kawamura et al. 1999). Furthermore, the nematode Caenorhabditis elegans and the zebra fish Danio rerio have multiple putative YKL-40-like proteins (18%-30% sequence identity).

YKL-40 gene

In 1997 the human gene encoding YKL-40 was isolated (Rehli et al. 1997). It is assigned to chromosome 1q31-q32 and consists of 10 exons and spans about 8 kilobases of genomic DNA. Recently the transcriptional regulation of YKL-40 during human macrophage differentiation has been described (Rehli et al. 2003). There are probably two independent transcription start sites and the promoter sequence contains binding sites for several known factors and specific binding of nuclear PU.1, Sp1, Sp3, USF, AML-1 and C/EBP proteins. It was further found that the Sp1-family transcription factors seem to have a predominating role in controlling YKL-40 promoter activity. It was also suggested that the YKL-40 gene in monocytes is in an inactive or unstable, yet primed state, which may require additional events (e.g. nucleosome remodeling) that may be initiated by additional elements upstream or downstream of the promoter (Rehli et al. 2003). The genes of the other human "chitinase-like proteins" known so far are also located on chromosome 1. The gene for chitotriosidase (Boot et al. 1998) is located on 1q31-1q32. The genes coding for YKL-39 (GenBank accession number U58514), OGP (Takahashi et al. 2000, GenBank accession numbers U58001-U58010), TSA1902 (Saito et al. 1999), and AMCase (Boot et al. 2001) are located on 1p13. The mouse YM1/ECF-L gene (Chang et al. 2001) is located on mouse chromosome 3, a chromosome that corresponds to human chromosome 1.

YKL-40 structure

The crystallographic three-dimensional structures of human YKL-40 (Fusetti et al. 2003; Houston et al. 2003) and goat YKL-40 (Mohanty et al. 2003) display the typical fold of family 18 glycosyl hydrolases (Henrissat et al. 1997). The structure is divided into two globular domains: a big core domain which consists of a (β/α)8 domain structure with a triose-phosphate isomerase (TIM) barrel fold, and a small α / β domain composed of five antiparallel β -strands and one α -helix that is inserted in the loop between strand β 7 and helix α 7 of the TIM barrel. This gives the active site of YKL-40 a groove-like character. YKL-40 is a lectin and bound carbohydrates are not hydrolyzed as discussed in detail below. A 43 Å long carbohydrate binding cleft is present at the C-terminal side of the β -strands in the ( β / α ) 8 barrel. The crystal structure of YKL-40 is similar in many aspects to the crystal structure of human chitotriosidase (Fusetti et al. 2002), mouse YM1 (Sun et al. 2001), Drosophila melanogaster IDGF-2 (Varela et al. 2002) and to other members of the glycosyl hydrolase family 18 (Coulson 1994; Reardon et al. 1995), but several major structural changes are also found. Family 18 chitinases contain a sequence motif DxxDxDxE which lies on strand β 4. The glutamic acid (E) is the catalytic acid, which pronates the glycosidic bond. The neighboring aspartic acid (D) plays a key role in orienting the N-acetyl group of the -1 sugar for nucleophilic attack on the anomeric carbon, and stabilizes the subsequently formed oxazolium ion intermediate (Van Aalten et al. 2001). In human YKL-40 there is a mutation of the catalytic glutamic acid to leucine (L, residue 140) and a mutation of the catalytic aspartic acid to alanine (A, residue 138). Both mutations appear to rule out a hydrolase activity for YKL-40. Although YKL-40 is not a chitinase, human YKL-40 binds chitin of different lengths and in a similar fashion as seen in Family 18 chitinases, and nine sugar-binding subsites are found in the 43 Å groove (Fusetti et al. 2003). The presence of chitin fragments in the binding groove does not cause drastic conformational changes in the protein (Fusetti et al. 2003). YKL-40 is N-glycosylated at asparagine (Asn, residue 60) and 2 β (1,4)-linked GlcNAc residues are visible in the electron density. Glycosylation is a unique feature of YKL-40 structure as the residue corresponding Asn (residue 60) does not exist in chitinases and is mutated to proline in other "mammalian chitinase-like proteins". YKL-40 binds heparin (Shackelton et al. 1995) and amino acid sequence analysis reveals that YKL-40 contains one heparin-binding motif (GRRDKQH, residue 143-149). This putative heparin-binding site is located in a surface loop (Fusetti et al. 2003). However, soaking of YKL-40 crystals or co-crystallization in the presence of fully sulfated heparin did not result in evidence of binding at this site. It has been suggested that heparan sulfate is a more likely ligand of YKL-40 (Johansen et al. 1997; Fusetti et al. 2003), and unsulfated fragments of heparan sulfate can be accommodated in the binding groove of YKL-40 (Fusetti et al. 2003). Amino acid sequence analysis reveals that YKL-40 contains two potential hyaluronan-binding sites on the external face of the folded protein (Malinda et al. 1999), but this has not been evaluated in crystallization studies. YKL-40 contains five cysteins and four are involved in two disulfide bridges (C 26 -C 51 and C 300 -C 364 ) (Fusetti et al. 2003) and conserved in all "mammalian chitinase-like proteins" (Sun et al. 2001; Fusetti et al. 2002; Mohanty et al. 2003). The free Cxx in YKL-40 is located in a tightly packed hydrophobic pocket. Three conserved cis peptides are present.

YKL-40 expression in non-malignant cells

Several different cell types of ectoderm, mesoderm, and endoderm origin express YKL-40 mRNA and protein in vitro and in vivo under specific conditions.

Macrophages

YKL-40 mRNA expression in vitro is absent in normal human monocytes but strongly induced during the late stages of human macrophage differentiation (Krause et al. 1996; Rehli et al. 1997; Renkema et al. 1998; Rehli et al. 2003) and by treatment of monocytes and the human monocytic cell line THP-1 with phorbol myristate acetate (PMA induces differentiation of monocytes into an adherent macrophage-like cell type) (Kirkpatrick et al. 1997; Rehli et al. 2003). Serial analysis of gene expression (SAGE) demonstrated 288 fold increased YKL-40 transcripts in monocytes stimulated with granulocyte-macrophage colony-stimulating factor (GM-CSF), a 182 fold increase in YKL-40 after stimulation with M-CSF and a 31 fold increase in YKL-40 transcripts in lipopolysaccharide stimulated monocytes (Hashimoto et al. 1999a; Suzuki et al. 2000). No YKL-40 expression was found in human monocytes or dendritic cells (Hashimoto et al. 1999b). In vivo YKL-40 mRNA and protein expression are found by a subpopulation of macrophages in different tissues with inflammation and extracellular matrix (ECM) remodeling: 1) macrophages in inflamed synovial membranes from patients with rheumatoid arthritis (RA), osteoarthritis (OA) or ankylosing spondylitis (AS) express YKL-40 mRNA and protein (Kirkpatrick et al. 1997; Baeten et al. 2000; Volck et al. 2001); 2) macrophages in atherosclerotic plaques express YKL-40 mRNA, particularly macrophages that had infiltrated deeper in the lesion, and the highest expression of YKL-40 is found in macrophages in the early lesion of atherosclerosis (Boot et al. 1999); 3) macrophages and giant cells located in the media of arteritic vessels of patients with giant cell arteritis (GCA) express YKL-40 protein (Johansen et al. 1999a, V); 4) giant cells in the sarcoid lesions of patients with pulmonary sarcoidosis (Johansen et al. 2005b) express YKL-40 protein; and 5) peritumoral macrophages in biopsies from small cell lung cancer express YKL-40 mRNA (Junker et al. 2005a). Another "mammalian chitinase-like protein", chitotriosidase, is also produced by activated macrophages but not by the same sub-population as YKL-40 (Boot et al. 1999).

A Drosophila melanogaster cell line exhibiting macrophage-like properties secretes a closely related protein to YKL-40 named DS47 (Kirkpatrick et al. 1995). This protein is expressed during the entire Drosophila melanogaster life cycle. In the larvae the DS47 message is found in the fat body (an organ that is somewhat analogous to the human liver) and by hemocytes and is secreted into the hemolymph. The IDGFs (proteins with amino acid sequence identity to YKL-40) are secreted by Drosophila yolk cells, the fat body and the imaginal disc (cells with macrophage-like properties) (Kawamura et al. 1999).

Using flow cytometry Baeten et al. (2000) showed that RA patients have YKL-40 positive (+) peripheral blood mononuclear cells (PBMC) and that these cells are CD16+ and have a dim expression of CD14. The CD14+,CD16+ monocyte phenotype can differentiate from classic CD14++ monocytes by maturation in vitro and resembles the monocyte population described by Ziegler-Heitbrock (1996), but their physiological role remains to be determined. The CD14+,CD16+ monocytes are increased in numbers in patients with RA (Baeten et al. 2000), sepsis (Fingerle et al. 1993), tuberculosis (Vanham et al. 1996) and solid tumors (Saleh et al. 1995). These monocytes are believed to be a more mature version of monocytes with properties of tissue macrophages, probably of pro-inflammatory type. They have a similar antigen-presenting potential as macrophages and produce proinflammatory cytokines, but produce little or no anti-inflammatory cytokines. They have a low capacity for phagocytosis and reactive oxygen production, and a high expression of major histocompatibility complex (MHC) class II antigens and adhesion molecules (Thieblemont et al. 1995; Frankenberger et al. 1996; Ziegler-Heitbrock et al. 1996).

The present studies show that YKL-40 is a phylogenetically highly conserved protein secreted by macrophages, and the expression of YKL-40 seems to be restricted to small, unique groups of macrophages exemplifying the phenotypic variation among macrophages.

Neutrophil granulocytes

Neutrophil granulocytes share a common progenitor cell with macrophages and neutrophil precursors begin to synthesize YKL-40 at the myelocyte-metamyelocyte stage (Volck et al. 1998). YKL-40 is stored in the specific granules of neutrophils and released after full activation of the neutrophils (Volck et al. 1998; Boussac et al. 2000). Chitotriosidase and YM1 are also neutrophil granule proteins but their exact subcellular localizations are unknown (Boussac et al. 2000; Harbord et al. 2002).

Chondrocytes

Hakala et al. reported in 1993 that YKL-40 mRNA expression is high in cartilage from RA patients and undetectable in normal cartilage. Cartilage explant- or monolayer chondrocyte cultures isolated from RA cartilage (Hakala et al. 1993) and OA cartilage (Johansen et al. 2001c, VII) secrete YKL-40 in vitro. Monolayer cultures of chondrocytes freshly isolated from normal cartilage secrete low levels of YKL-40, but this basal production of YKL-40 increase more than 300 fold in first- and second-passage of chondrocyte cultures (Johansen et al. 2001c,VII). Microarray cDNA analysis have demonstrated that YKL-40 gene expression is up-regulated in dedifferentiated human fetal chondrocytes compared to chondrocytes maintained in a differentiated state (Stokes et al. 2002). Chondrocytes cultured in monolayer become dedifferentiated, acquiring a fibroblast-like appearance and changing their pattern of gene expression from one that express chondrocyte-specific genes to one that resembles a fibroblastic or chondroprogenitor-like pattern. In vitro re-differentiation of dedifferentiated chondrocytes investigated by cDNA analysis show increased YKL-40 expression as does in vitro chondrogenesis (Imabayashi et al. 2003), indicating that YKL-40 is a differentiation marker in chondrocytes. In-situ hybridization analysis have shown that YKL-40 mRNA is undetectable in chondrocytes from normal articular cartilage but is expressed in moderate to high levels in chondrocytes located in the superficial zone of articular cartilage with mild OA. In advanced OA chondrocytes located in both the superficial, middle and deep layer express YKL-40 and this increase with the extent of tissue damage (Connor et al. 2000). High YKL-40 mRNA expression is also found in chondrocytes in the pre-secondary ossification center of developing fetal cartilage whereas chondrocytes located in the growth plate and mineralized cartilage have lower YKL-40 expression (Connor et al. 2000). Immunohistochemical analysis have shown YKL-40 protein expression in chondrocytes located in both the superficial and middle layer of cartilage biopsies from RA and OA patients (Volck et al. 1999, 2001; Johansen et al. 2001c, VII; Kawasaki et al. 2001). The intracellular presence of YKL-40 in chondrocytes is shown in the Golgi apparatus and the endoplasmic reticulum (Johansen et al. 2001c, VII). YKL-39, another "mammalian chitinase-like protein", is also secreted in vitro by monolayer cultures of human chondrocytes isolated from normal cartilage but is a less abundant protein compared to YKL-40 (Hu et al. 1996).

Fibroblast-like synovial cells (synoviocytes)

Synovial cells obtained from the synovial membrane of RA patients at time of joint replacement secrete YKL-40 in vitro (Nyirkos et al. 1990), and YKL-40 mRNA expression is found in inflammed synovial membrane from RA patients but not in non-inflamed synovial membrane (Hakala et al. 1993). Dasuri et al. (2004) studied proteins of fibroblast-like synovial cells from RA patients, using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, and found that YKL-40 is a major cellular protein in these cells.

Bone cells

Monolayer cultures of osteoblasts from adult and fetal bone do not secrete YKL-40 in vitro (Johansen et al. 1992). However, YKL-40 mRNA expression is found in end-stage osteoblasts in osteophytic tissue and in primary osteocytes and osteoblasts at sites of endochondral and intramembranous bone formation. YKL-40 mRNA expression is low to moderate in osteoid-forming and proliferating osteoblasts and undetectable in fully mature osteocytes and osteoclasts, indicating a maturation stage-dependent expression of YKL-40 in osteoblasts and osteoclasts (Connor et al. 2000). It is not known if activated osteoclasts express YKL-40.

Vascular smooth muscle cells

YKL-40 is synthesized in vitro by vascular smooth muscle cells isolated from explants of swine thoracic aorta during the time of transition from a proliferating monolayer culture to a non-proliferating differentiated multilayer culture (Millis et al. 1985; Millis et al. 1986). YKL-40 secretion continues as the cells reorganize and form multicellular nodules in which cells reexpress markers of differentiated vascular smooth muscle cells (Millis et al. 1985; Shackelton et al. 1995; Malinda et al. 1999). This in vitro nodule forming process mimics some of the characteristics of the in vivo changes that occur in vascular wall smooth muscle cells following injury where media smooth muscle cells dedifferentiate, migrate, and contribute to the process of restenosis and neointima formation (Schwartz 1997). Immunohistochemical analysis show YKL-40 protein expression in human smooth muscle cells in adventitial vessels (Johansen et al. 1999a, V) and atherosclerotic plaques (Nishikawa et al. 2003).

Liver cells

Hakala et al. reported in 1993 that a strong YKL-40 mRNA expression was found in human liver. However, this could not be reproduced by Hu et al. (1996). The liver tissue used in the study by Hakala et al. (1993) may have originated from a fibrotic liver. Immunohistochemical studies of liver biopsies have shown YKL-40 protein expression in areas of the liver with fibrosis and no expression in hepatocytes (Johansen et al. 1997; Johansen et al. 2000a, VI). Suppression subtractive hybridization analysis and RT-PCR have found that YKL-40 is one of the most overexpressed proteins in cirrhotic liver tissue caused by hepatitis C virus (HCV) (Shackel et al. 2003). The hepatic stellate cell (HSC), the principal effector cell in liver fibrogenesis (Friedman 2000), express YKL-40 mRNA in vitro but YKL-40 protein in conditioned media from human HSC have not yet been detected (E. Efsen, manuscript in preparation).

Mammary epithelial cells

YKL-40 in mice is called the "breast regression protein (Brp-39)" (Morrison et al. 1994) because it is induced in mammary epithelial cells a few days after weaning. YKL-40 is not detectable in milk during lactation but is isolated from the whey protein secretions of bovine mammary secretions during the nonlactating period after weaning (Rejman et al. 1988) and in bovine colostrum (Yamada et al. 2002).

Malignant cells

Se Chapter 5.

Other cells/tissues

cDNA microarray analysis have demonstrated that 1) hippocampus tissue from patients with schizophrenia have elevated YKL-40 expression compared to control hippocampus tissue (Chung et al. 2003); 2) Helicobacter- infected murine stomachs have increased YKL-40 expression compared to uninfected stomachs (Mueller et al. 2003); 3) YKL-40 expression in ovariectomiced murine chorioretinal tissue is downregulated by 17- β -estradiol (Rakic et al. 2003); and 4) YKL-40 is expressed in normal human neural retina and retinal pigment epithelium-choroid complex, and upregulated in pathological human exudative age-related macular degeneration and experimental murine choroidal neovascular membranes (Sharon et al. 2002; Rakic et al. 2003). Suppression subtractive hybridization analysis of genes from human mesothelial cells, obtained from benign effusions, that differentiate into a fibroblastic morphology show more than 20 fold overexpression of YKL-40 (Sun et al. 2004).

Biologic activities of YKL-40

YKL-40 is a secreted protein suggesting that its sites of actions are most likely to be extracellular. Specific cell-surface or soluble receptors for YKL-40 have not yet been identified. The biological function of YKL-40 is not yet clear, but several possible functions have been proposed:

Growth properties

In vitro studies have shown that YKL-40 in physiological concentrations increases proliferation of guinea pig chondrocytes, rabbit chondrocytes and synovial cells, and that YKL-40 increases proteoglycan synthesis of guinea pig and rabbit chondrocytes (De Ceuninck et al. 2001a). Recklies et al. (2002) found that YKL-40 increases growth rates of three fibroblastic cell lines derived from human osteoarthritic synovium, fetal lung and adult skin. The magnitude of the response of YKL-40 stimulation on synovial cells and skin fibroblasts on incorporation of [ 3 H]thymidine into cellular DNA is similar to that elicited by the insulin-like growth factor-1 (IGF-1), and YKL-40 and IGF-1 work synergistically in stimulating growth of the fibroblasts. YKL-40 initiates mitogen-activated protein (MAP) kinase and PI-3K signaling cascades in fibroblasts leading to phosphorylation of both the extracellular signal-regulated kinase (ERK)-1/2 MAP kinase and protein kinase B (AKT)-mediated signaling cascades (Recklies et al. 2002), which are associated with the control of mitogenesis. This suggests a role of YKL-40 as an anti-apoptotic protein. The PI-3K pathway, and in particular the phosphorylation of AKT, is strongly associated with cell survival (Bakkenist et al. 2004; Downward 2004; Mitsiades et al. 2004). Identity of cellular receptors mediating the biological effects of YKL-40 are currently not known, but the activation of cytoplasmic signal-transduction pathways suggests that YKL-40 interacts with one or several signaling components on the plasma membrane. Recently, Ling et al. (2004) showed that stimulation of human articular chondrocytes or skin fibroblasts with interleukin 1 (IL-1) or tumor necrosis factor alfa (TNF α ) in the presence of YKL-40 results in reduction of both p38 and SAPK/JNK phosphorylation, and that YKL-40 suppresses the cytokine-induced secretion of metalloproteinase (MMP)-1, MMP-3 and MMP-13 and the chemokine IL-8. The suppressive effect of YKL-40 is dependent on kinase activity, and treatment of articular chondrocytes and skin fibroblasts with YKL-40 results in AKT-mediated serine/threonine phospholyration of the apoptosis signal-regulator kinase, ASK1. It was suggested that YKL-40 elicits an anti-catabolic effect preserving ECM during tissue remodeling/destruction (Ling et al. 2004).

YKL-40 in mice is called the "breast regression protein (Brp-39)" (Morrison et al. 1994) because it is induced in mammary epithelial cells a few days after weaning. Mammary involution involves programmed cell death, and it has been suggested that YKL-40 utilizes a chitin oligosaccharide binding ability while participating in the various signal transduction pathways that lead to apoptosis of the regressing cells. Mohanty et al. (2003) hypothesized that YKL-40 is a protective signaling factor that determines which cells are to survive the drastic tissue remodeling that occurs during involution.

YKL-40 acts as a chemoattractant for human umbilical vein endothelial cells and stimulates migration of these cells at a level comparable to that achieved with the endothelial cell chemoattractant basic fibroblast growth factor (bFGF) (Malinda et al. 1999). YKL-40 modulates vascular endothelial cell morphology by promoting the formation of branching tubules, indicating that YKL-40 may function in angiogenesis by stimulating the migration and reorganization of vascular endothelial cells (Malinda et al. 1999). Furthermore, YKL-40 promotes vascular smooth muscle cell attachment, spreading and migration, suggesting that YKL-40 has a role in the process of atherosclerotic plaque formation where smooth muscle cells are induced to migrate through the intima in response to exogenous signals (Nishikawa et al. 2003).

In contrast to many other cell types, chondrocytes and the human osteosarcoma cell line MG63 can be maintained in unsupplemented culture medium for about 14 days without any loss of viability (Johansen et al. 1992; Johansen et al. 2001c, VII). Furthermore, the persistence of YKL-40 secretion from these cells and swine vascular smooth muscle cells (Millis et al. 1985) is not dependent on the presence of serum. The lack of a requirement for growth factor supplementation may be due to their own production of large amounts of YKL-40 when cultured in serum free media, indicating that YKL-40 may play a role in cell growth and survival.

YKL-40 may also has a functional role in embryonic development. In the developing mouse heart expression of YKL-40 coincides with morphological changes involving cell migration, altered cell adhesion and remodeling suggesting a role for YKL-40 in cardiac morphogenesis consistent with its established activities in vitro of promoting cell migration and adhesion (Nishikawa et al. 2003).

Several other "mammalian chitinase-like proteins", human AMCase/rat iSBLP 58 , human ECF-L/mouseYm1, Drosophila IDGFs, also have growth factor activity in vitro (Guoping et al. 1997; Kawamura et al. 1999; Owhashi et al. 2000), whereas human chitotriosidase has no mitogenic effect on skin, fetal lung or synovial fibroblasts (Recklies et al. 2002). It has been suggested that Drosophila IDGFs may have evolved from chitinases to acquire new functions as growth factors, interacting with cell surface glycoproteins implicated in growth-promoting processes such as the Drosophila insulin receptor (Varela et al. 2002).

It has been suggested that YKL-40 may be a differentiation marker since elevated YKL-40 expression is found when monocytes differentiate to macrophages (Krause et al. 1996; Rehli et al. 1997; Renkema et al. 1998; Rehli et al. 2003), when mesothelial cells differentiate into a fibroblast like morphology (Sun et al. 2004), when vascular smooth muscle cells differentiate (Millis et al. 1985; Shackelton et al. 1995; Malinda et al. 1999), and when chondrocytes differentiate to fibroblast like cells or re-differentiate to chondrocytes (Stokes et al. 2002; Imabayashi et al. 2003).

Heparin binding properties

YKL-40 contains a single putative heparin binding site (location 144-147) (Malinda et al. 1999, Nishikawa et al. 2003) and binds heparin with an affinity greater than fibronectin (Millis et al. 1985; Millis et al. 1986; Shackelton et al. 1995). YKL-40 may interact with heparin-like molecules in the ECM or on the cell surfaces. Fusetti et al. (2003) have shown using crystallization methods that heparan sulfate and not heparin is a more likely physiological ligand of YKL-40. The physiological role of heparan sulfate proteoglycans are highly diversified, including cell adhesion, proliferation, migration, differentiation, growth factor and cytokine action, tissue morphogenesis/organogenesis, tissue remodeling and wound healing (Iozzo et al. 1994; SundarRaj et al. 1995). YKL-40 may function by interacting with components of the cell surface or the ECM and one physiologic ligand for YKL-40 could be perlecan (a heparan sulfate proteoglycan) which is a component of basement membranes and expressed in the ECM of many tissues including cartilage, liver, and cancer. Proteins anchored on glycosaminoglycan side chains of heparan sulfate proteoglycans serve a variety of functional purposes, from simple immobilization or protection against degradation to modulation of distinct biological activities (Lindahl et al. 1998; Perrimon et al. 2000). Transient and selective expression of heparan sulfate proteoglycans is elucidated as to deliver growth factors (e.g. FGF, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF)) to their appropriate receptors on fibroblasts or endothelial cells for signaling new tissue growth during the repair processes (Clasper et al. 1999).

Chitin binding properties

YKL-40 has high amino acid sequence homology to bacterial chitinases (Hakala et al. 1993; Johansen et al. 1993 I) and strong binding affinity for chitin (Hakala et al. 1993; Renkema et al. 1998; Houston et al. 2003), but YKL-40 lacks chitinase activity (Hakala et al. 1993; Renkema et al. 1998). Chitooligosaccharides bind to YKL-40 with μ M affinity (Houston et al. 2003), and oligomeric chitin could be a physiological ligand for YKL-40, although binding of other carbohydrate polymers cannot be excluded. The chitinase activity is dependent on aspartic acid (D) and glutamic acid (E) at the end of the conserved catalytic center DxxDxDxE sequence motif (Watanabe et al. 1993; Watanabe et al. 1994; van Aalten et al. 2001; Bokma et al. 2002). The essential aspartic acid is conserved in YKL-40, Drosophila DS47 and IDGF1, and in all the "mammalian chitinase-like proteins" except in mouse Ym1, where it is replaced by asparagine. The essential glutamic acid is replaced by leucine in YKL-40 and OGP, by isoleucine in YKL-39, and by glutamine in mouse Ym1, Drosophila DS47 and IDGF1. None of these proteins have chitinase activity. Of the known "mammalian chitinase-like proteins" only chitotriosidase (a protein expressed by macrophages and neutrophils) (Hollack et al. 1994; Boot et al. 1995; Boussac et al. 2000) and AMCase (a protein expressed in the gastrointestinal tract and lung) (Boot et al. 2001) have chitinase activity and both have glutamic acid in the essential position. A single amino acid substitution in the catalytic domain of chitotriosidase, generating the same amino acid sequence as in YKL-40, is followed by the loss of hydrolytic activity and retains the capacity to bind chitin (Renkema et al. 1998). In plants chitinases are believed to form part of innate immune system important for host defense against invading pathogenic bacteria and fungi and plants produce high amounts of chitinases under conditions of stress (Collinge et al. 1993; Sahai et al. 1993). It has been suggested that human chitotriosidase and AMCase play a role in host defense through degradation of chitin-containing cell walls of fungal pathogens (Boot et al. 2001). YKL-40 is secreted by macrophages and neutrophils, which serve in the primary defense mechanisms against invading pathogens. It has therefore been suggested that YKL-40 could act as a opsonin with a role in the immune response (Renkema et al. 1998), and that YKL-40 could act as a chitin sensor, switching on innate defenses, helping to direct macrophages to the site of invasion and to regulate the inflammatory response as a consequence of infection (Houston et al. 2003). Another of the "mammalian chitinase-like proteins", Ym1/ECF-L, has been proposed to have a role in directing components of the immune system to the site of nematode infections (Owashi et al. 2000).

Chitinases catalyze the hydrolysis of β -1,4-N-acetylglucoside linkages in chitin, a homopolysaccharide that consists of repeated N-acetyl- β -(1,4-linked) D-glucosamine (2-deoxy-2acetamino-D-glucose). Chitin is the principal structural component of the cell walls of plants, algae, fungi and bacteria, the microfilarial sheath of parasitic nematodes, of the shells or cuticles of arthropods, the lining of guts of many insects, in nematodes, mollusk, in worms and in the exoskeleton of fish and vertebrates (Flach et al. 1992; Araujo et al. 1993; Debone et al. 1994; Shahabuddin 1994). Although chitin is not found in mammals, YKL-40 may interact with an so far unknown endogenous compounds with chitin-like motifs that may exist in mammals. It has been found in vertebrates in an embryonic stage that short chito-oligosaccharides are used as primers for the synthesis of hyaluronan (Meyer et al. 1996; Semino et al. 1996; Varki 1996).

Effect on hyaluronan synthesis

Hyaluronan is a linear polysaccharide composed of repeating disaccharide units of N-acetyl glucosamine and D-glucoronate linked together by alternating β (1,4) and β (1,3) glycosidic bonds. Hyaluronan is located in the ECM of many tissues and is synthesized by articular chondrocytes, synovial cells in the inflamed synovial membrane, smooth muscle cells in injured vessels, hepatic stellate cells in liver fibrosis, fibroblasts in skin tissue and by cancer cells. Hyaluronan has multiple physiological roles and has been connected with embryogenesis, morphogenetic processes, cell proliferation and tissue remodeling and is involved in acute and chronic inflammatory processes (Laurent 1998; Lee et al. 2000). The expression of YKL-40 is related to similar events as hyaluronan (see Chapter 4 and 5) and the function of YKL-40 may be linked to the functions of hyaluronan. It is not known if YKL-40 binds hyaluronan, but YKL-40 has two potential hyaluronan binding motifs (location 147-155 and 369-377) (Malinda et al. 1999; Nishikawa et al. 2003). YKL-40 may recognize hyaluronan, or its precursor, as a substrate in the ECM and interfere with its synthesis, which could affect local hyaluronan levels and consequently influence the extent of cell adhesion and migration during the tissue remodeling processes that take place during inflammation, fibrosis, atherogenesis and metastasis.

Regulation of YKL-40

Effect of different extracellular matrix (ECM)

Several studies indicate that changes in ECM are related to changes in YKL-40 synthesis. Changes in the ECM environment of chondrocytes seem to affect YKL-40 production by these cells. Chondrocytes propagated under culture conditions undergo phenotypic changes both in morphology (i.e. the loss of the chondrocyte spherical shape and the acquisition of an elongated fibroblast-like morphology) and in gene expression pattern. The morphologic alterations of the chondrocytes are accompanied by profound biochemical changes, including loss of the cartilage-specific phenotype, as evidenced by an arrest of the synthesis of the cartilage-specific collagens (types II, IX, and XI) and proteoglycans (aggrecan), initiation of synthesis of the interstitial collagens (types I, III, and V), and increase in the synthesis of fibroblast-type proteoglycans at the expense of aggrecan. The chondrocyte phenotype can be re-expressed in the cells by culturing them in suspension, in agarose, with alginate beads, or on a hydrogel substrate (Benya et al. 1982; Bonventure et al. 1994; Haüselmann et al. 1992 and 1994; Freed et al. 1993; Reginato et al. 1994). These changes in the biosynthetic profile of dedifferentiated chondrocytes resemble some of the phenotypic changes displayed by OA chondrocytes, and the matrix they produce is similar to that synthesized by chondroprogenitor cells (Benya et al. 1978; Kosher et al. 1986; Aigner et al. 1993, 1997 and 1999). Microarray gene expression analysis of human fetal chondrocytes cultured either under conditions that allow them to preserve their differentiated phenotype or under conditions that lead to their dedifferentiation show that YKL-40 was overexpressed 4.4 fold in dedifferentiated human fetal chondrocytes compared to differentiated chondrocytes (Stokes et al. 2002). Normal cartilage explant cultures produce low levels of YKL-40 during the first days of culture but after a few days YKL-40 secretion increases. Stimulation of YKL-40 production is also generated by the trauma of cartilage resection and by removal of chondrocytes from their native ECM environment. Freshly isolated chondrocytes from normal cartilage do not secrete YKL-40 during the fist days of monolayer culture, but in first-passage monolayer cultures the cells produce > 300-fold higher levels of YKL-40 compared to primary chondrocyte cultures. If chondrocytes are cultured as monolayer or in suspension with methacrylate, the cells first have to make cell-to-cell contact and then they produce an ECM. Chondrocytes cultured in these two systems secrete large amounts of YKL-40 and the production is prevented if the chondrocytes are cultured in alginate, where the cells already are surrounded by an ECM (Johansen et al. 2001c VII).

The production of YKL-40 is also increased many fold after monolayer cultures of smooth muscle cells form nodules. This YKL-40 synthesis is not the result of the absence of cell proliferation, but is closely linked to nodule formation (Millis et al. 1985). The expression of YKL-40 mRNA from mouse mammary tissue at different stages of functional differentiation shows that YKL-40 is expressed at very low levels prior to and during pregnancy and lactation, but its expression increases many fold during mammal gland involution (Morrison et al. 1994) which is characterized by increased tissue remodeling.

Effect of cytokines and growth factors

Regulatory studies of YKL-40 expression by cytokine and growth factors are sparse. IL-1 β inhibited YKL-40 mRNA expression and secretion by human monolayer chondrocyte and cartilage explant cultures (Johansen et al. 2001c, VII) but has no effect on YKL-40 secretion by guinea pig chondrocyte cultures (De Ceuninck et al. 1998). Transforming growth factor β (TGF β ) reduces YKL-40 mRNA expression in human chondrocytes (Hakala et al. 1993; Johansen et al. 2001c, VII), as well as the synthesis of YKL-40 from human chondrocytes (Johansen et al. 2001c, VII) and guinea pig chondrocytes (De Ceuninck et al. 1998). YKL-40 secretion by freshly isolated chondrocytes (Johansen et al. 2001c, VII) is stimulated by IL-6 (a cytokine with important roles in the inflammatory process (Xing et al. 1998)), IL-17 (a proinflammatory cytokine with a role in joint inflammation and acts in synergy with IL-1 β and tumor necrosis factor α (TNF α ) (Miossec 2003)), and IL-18 (a cytokine contributing to cartilage degradation (Olee et al. 1999)). IGF-I and IGF-II stimulate YKL-40 synthesis by guinea pig chondrocytes (De Ceuninck et al. 1998) but not by human chondrocytes (Hakala et al. 1993; Johansen et al. 2001c, VII). The controversy with the effectiveness of IL-1 β and IGF-I on human and guinea pig chondrocytes may rest on differences in the species, donor age, doses or tissue conditions. TNF α , platelet-derived growth factor (PDGF), bFGF, 1,25(OH) 2 D 2 , dexamethasone and serum have no effect on YKL-40 production by monolayer chondrocyte and cartilage explant cultures (De Ceuninck et al. 1998; Johansen et al. 2001c, VII). The regulation in vitro of YKL-40 secretion by human synovial cells was not influenced by IL-1 β and TNF α (Nyirkos et al. 1990).

Conclusions and future perspectives

YKL-40 can be categorized as a member of the glycosyl hydrolase family 18 that includes at least eight "mammalian chitinase-like proteins". The conservation of exon-intron boundaries of the genes of the "mammalian chitinase-like proteins" and their chromosomal location, amino acid sequence homology and structural similarities suggest that the genes of these members of the "mammalian chitinase-like protein family" have evolved from chitinases to acquire new properties. YKL-40 is a secreted protein produced in humans by activated macrophages and neutrophils in different tissues with inflammation and increased remodeling of the ECM, by arthritic or injured chondrocytes, by fibroblast-like synovial cells, by vascular smooth muscle cells, and probably by hepatic stellate cells. The complete in vivo biological functions of YKL-40 remains to be established, but it may have a function in inflammation and remodeling of the ECM. In vitro studies have demonstrated that YKL-40 exerts growth factor properties on fibroblasts, chondrocytes, fibroblast-like synovial cells, and endothelial cells, and promotes vascular smooth muscle cell attachment, spreading and migration. It has been hypothesized that YKL-40 protects cells from undergoing apoptosis. YKL-40 may also have a role in hyaluronan synthesis and, due to its heparin/heparan sulfate binding properties, its function could be linked to changes in the ECM. The elucidation of the biological function of YKL-40 is an important objective of future studies and YKL-40 transgenic and knock-out mice will hopefully be developed. Also research on YKL-40 potential receptor(s), the regulation of YKL-40 expression, and the evolutionary relationship of the different members of the "mammalian chitinase-like protein family" could give insights into the physiological role of YKL-40 and its family members.

Microarray cDNA analysis

Several studies have evaluated YKL-40 gene expression using microarray gene analysis. Different human tissues and cells are tested: monocytes and macrophages (Hashimoto et al. 1999a; Suzuki et al. 2000), fetal chondrocytes (Stokes et al. 2002), gliomas (Lal et al. 1999; Markert et al. 2001; Tanwar et al. 2002), thyroid carcinomas (Huang et al. 2001), extraskeletal myxoid chondrosarcoma (Sjögren et al. 2003), hippocampus tissue (Chung et al. 2003), Helicobacter- infected murine stomachs (Mueller et al. 2003) and murine ovariectomiced chorioretinal tissue (Sharon et al. 2002; Rakic et al. 2003).

In situ hybridization

In situ hybridization studies have evaluated YKL-40 mRNA expression in frozen and formalin-fixed paraffin-embedded tissues in different human tissues: synovial membrane from RA patients (Kirkpatrick et al. 1997), normal and atherosclerotic coronary arteries and aorta (Boot et al. 1999), cartilage and osteophytic tissue from OA patients and healthy adults and from normal and fetal bone (Connor et al. 2000).

Immunohistochemical analysis

Several immunohistochemical procedures for the detection of YKL-40 protein expression in biopsies of human tissues have been described using well known immunohistochemical methods for frozen or formalin-fixed paraffin-embedded tissues. An affinity purified rabbit antibody against human YKL-40 (Johansen et al. 1997, 1999a V, 2000a VI; Volck et al. 1998, 1999, 2001; Kawashaki et al. 2001) or a mouse monoclonal antibody against human YKL-40 (Baeten et al. 2000; Johansen et al. 2001c VII) were used as primary antibody. Different human tissues are evaluated: liver (Johansen et al. 1997, 2000a VI), bone marrow (Volck et al. 1998), inflamed arteries (Johansen et al. 1999a V), cartilage (Volck et al. 1999, 2001; Johansen et al. 2001c VII; Kawashaki et al. 2001), synovial membrane (Baeten et al. 2000; Volck et al. 2001), peripheral blood mononuclear cells (Baeten et al. 2000) and atheroslerotic vessels (Nishikawa et al. 2003).

Radio- and enzyme-linked immunoassays
for the determination of YKL-40

Three immunoassays for the measurement of human YKL-40 in body fluids (serum, plasma, synovial fluid, cerebrospinal fluid) and conditioned human cell culture media are described in the literature (Johansen et al. 1993 I; Harvey et al. 1998; Vos et al. 2000b). The first human YKL-40 assay was a radioimmunoassay (RIA) using a rabbit polyclonal antibody against human YKL-40 (Johansen et al. 1993 I). The assay runs over two days, involves 20 hours incubation at room temperature and requires sample dilution. The sensitivity of the RIA was 10 μ g/l and the recovery 100.3%. The intraassay coefficient of variation (CV) was < 6.5%. The short term interassay CV (during a 5 months period) was < 12% (Johansen et al. 1993 I) and the long term interassay CV (during a 5 years period) was < 15% (personal observation). A two-site, sandwich-type enzyme-linked immunoassay (ELISA) for measurement of human YKL-40 was later developed and is commercially available from Quidel (CA, USA) (Harvey et al. 1998). This assay uses streptavidin-coated microplate wells, a biotinylated-Fab monoclonal mouse antibody against human YKL-40 (capture antibody) and an alkaline phosphatase-labeled polyclonal rabbit antibody against human YKL-40 (detection antibody). Bound enzyme activity is detected with p-nitrophenyl phosphate as substrate. The ELISA is finished within 4 hours and involves three 1-hour incubation steps, is carried out at room temperature and does not require sample dilution (only if the concentration of YKL-40 in the sample is very high). The sensitivity of the ELISA was 8 μ g/l and the recovery 102%. The intraassay CV was < 3.6%. The short term interassay CV (during a 11 days period) was < 3.7% (Harvey et al. 1998) and the long term interassay CV (during a 5 years period) is < 8.6% (personal observation).

The YKL-40 protein used for standards and antigen for antibody production in the RIA and ELISA and for tracer in the RIA was purified from serum-free conditioned medium of monolayer cultures of the human osteosarcoma cell line MG63 (Johansen et al. 1993 I; Harvey et al. 1998) by a modification of the heparin-affinity chromatography method described to purify YKL-40 (Rejman et al. 1988; Nyirkos et al. 1990). MG63 cells (obtained from the American Type Culture Collection, Rockville, MD) are easily cultured in serum free media and at confluence these cells secrete large amounts of YKL-40 (Johansen et al. 1992). Preparations of polyclonal or monoclonal antibodies were produced by routine procedures (Johansen et al. 1993 I; Harvey et al. 1998), but the specific epitopes of human YKL-40 recognized by these antibodies are unknown. Human YKL-40 concentrations in blood (serum or EDTA plasma), synovial fluid and cerebrospinal fluid (described in Chapter 3 and 4), and in conditioned medium of human cell cultures (Johansen et al. 2001c VII) can be determined using these methods. The YKL-40 ELISA is also useful for the measurement of serum YKL-40 levels in baboons (Mahanery et al. 1998) and cynomolgus macaques (Register et al. 2001). The YKL-40 RIA and ELISA can not detect mouse, rabbit, cow or swine YKL-40.

High correlation (Spearmans rho = 0.91, p < 0.0001) was found between the serum concentrations of YKL-40 determined by RIA and ELISA. The mean serum YKL-40 ELISA /YKL-40 RIA ratio was 0.479 calculated from 506 serum samples from 245 healthy adults, 112 RA patients, 37 OA patients and 112 patients with metastatic breast cancer (personal observation). The difference between serum YKL-40 levels using the two methods is probably explained by differences in the methods used to calculate YKL-40 protein concentration in the standards used in the two assays. The relative YKL-40 antigen recognition by the two assays was constant. In the following chapters and in the data presented in Figures 2-5 and Tables 1-4 the serum concentrations of YKL-40 determined by the YKL-40 RIA have been adjusted to YKL-40 ELISA results by multiplication with 0.479 for a better comparisons of the serum YKL-40 levels in the different studies.

Three other YKL-40 ELISAs have been developed but have only been used in a few clinical studies. Vos et al. (2000b) developed a human YKL-40 ELISA using plates coated with a mouse monoclonal antibody against human YKL-40 (capture antibody) and a horseradish peroxidase (HRP) labeled mouse monoclonal antibody against human YKL-40 (detection antibody). Rejman et al. (1989) developed an ELISA for YKL-40 determination in bovine mammary milk secretions during involution using plates coated with bovine YKL-40, a rabbit polyclonal antibody against bovine YKL-40 and HRP-technique. De Ceuninck et al. (2001b) developed an indirect competition ELISA for measurement of serum YKL-40 in guinea pigs using a polyclonal anti-guinea pig YKL-40 antibody produced in hens and extracted from the egg yolk. This assay can also determine rabbit YKL-40 but not rat or mice YKL-40.

Serum/plasma concentrations of YKL-40
in healthy subjects

The individual serum concentrations of YKL-40 in 245 healthy adults (aged 18-79 years) according to age is illustrated in Figure 1 . The subjects are described by Johansen et al. (1996a III) using the YKL-40 RIA method and their serum YKL-40 level was later determined by YKL-40 ELISA in 1997. No difference in serum YKL-40 was found between gender, but there was a relation between serum YKL-40 and age (rho = 0.45, p < 0.001) with the highest levels in the elderly. All subjects were healthy without symptoms of disease and were not taking medicine at the time of blood sampling. They had normal serum levels of creatinine, albumin, lactate dehydrogenase (LDH), aspartate aminotransferase, alkaline phosphatase and bilirubin. These subjects were not followed prospectively and it is not known if some later developed cancer that was not clinically detectable at the time of blood sampling. Aging is associated with low-grade inflammation (Bruunsgaard et al. 2001) and the increase in serum YKL-40 in elderly healthy subjects may be due to low-grade inflammation. Serum YKL-40 in 476 healthy children (aged 7-17 years) was similar to the level in healthy young adults and there was no change in serum YKL-40 during puberty (Johansen et al. 1996a III).

There is good agreement between serum concentrations of YKL-40 in the two largest studies of healthy subjects (Johansen et al. 1996a III; Harvey et al. 1998) with a median serum YKL-40 of 43 μ g/l (90% percentile = 95 μ g/l; 95% percentile = 124 μ g/l). The serum YKL-40 level in the study by Garnero et al. (2001) was higher, but these controls were older compared to the other studies of healthy subjects. In clinical studies of serum concentrations of YKL-40 in patients with different diseases it is important to compare serum YKL-40 in the patients with an age-corrected upper normal serum YKL-40 (e.g. 95 th percentile).

Haemodynamic investigations with catherization of the femoral artery and renal vein indicated that the kidney is the main site of YKL-40 disposal (Johansen et al. 1997). The plasma concentration of YKL-40 in the renal vein was significantly lower than in the femoral artery both in subjects with normal liver function and in patients with chronic liver disease. Furthermore, YKL-40 can be detected in urine (personal observation). In healthy subjects there was no correlation between serum concentrations of YKL-40 and creatinine (Johansen et al. 1996a III). Whereas patients with severe renal diseases (i.e. requiring hemodialysis or peritoneal dialysis) had significantly elevated serum YKL-40 compared to healthy subjects (personal observation).

Individual variation
in serum YKL-40 concentrations

There was no circadian variability in serum concentrations of YKL-40 in samples collected 7 times during the day from 16 healthy subjects (aged 32-66 years) and 21 patients with RA (aged 30-75 years). The long time CV in serum YKL-40 was 5% in 30 healthy women (aged 24-62 years) who had serum samples collected 5 times with seven days intervals and subsequently again after 3 years (Johansen et al. manuscript in preparation).

Stability of YKL-40 concentrations in blood
after venipuncture

Several factors must be considered when handling blood samples for the measurement of YKL-40. The time interval between drawing of blood and centrifugation of blood stored at room temperature must be less than 3 hours for serum and 8 hours for EDTA plasma samples. Otherwise significant and not disease related elevations of YKL-40 are found in the serum and EDTA plasma samples left on the clot for a longer time when compared with YKL-40 concentrations in serum and EDTA plasma samples centrifuged within 1 hour after venipuncture. If the blood was stored at 4 °C before centrifugation YKL-40 concentrations were stable in serum for 24 hours and in EDTA plasma for 72 hours (Høgdall et al. 2000b). Degranulation of neutrophils with release of YKL-40 from the specific granules is the most likely explanation for this time dependent increase in YKL-40 concentrations in serum and EDTA plasma. YKL-40 accumulated extracellularly in a time-dependent manner in standard erythrocyte components, and prestorage leukocyte depletion of whole blood prevented extracellular YKL-40 accumulation (Cintin et al. 2001). Repetitive freezing and thawing of serum samples up to 9 times had no effect on the serum YKL-40 (Johansen et al. 1993 I; Harvey et al. 1998; Høgdall et al. 2000b; De Ceuninck et al. 2001b; Vos et al. 2001b). YKL-40 concentrations in serum were stable in samples stored up to 5 days at room temperature (Johansen et al. 1993 I), up to 9 days at 4 °C (Harvey et al. 1998), and at -20 °C or -80 °C for at least 8 years (personal observation). YKL-40 concentrations in corresponding serum and EDTA plasma samples were correlated (rho = 0.98, p < 0.001), but YKL-40 was significantly higher in serum compared to EDTA plasma with a YKL-40 serum/EDTA plasma ratio of 1.4 (Johansen et al. 1993 I; Høgdall et al. 2000b). This is probably caused by a small release of YKL-40 from activated neutrophils during the coagulation process. In this thesis there will no discrimination between YKL-40 in serum and plasma samples, since the serum or plasma concentrations of YKL-40 in the patients were accordingly related to the serum or plasma concentrations of YKL-40 in healthy subjects.

Conclusions and future perspectives

The in-house YKL-40 RIA and the commercial YKL-40 ELISA are both satisfactory methods for measurement of serum concentrations of YKL-40 in terms of reliability, reproducibility and stability. YKL-40 is detectable in serum of apparently healthy subjects and increases with older age. Puberty, a physiological condition with increased remodeling of ECM, does not result in increased serum YKL-40 levels. Most of the circulating YKL-40 in healthy subjects probably originates from activated macrophages and neutrophils. The high serum YKL-40 in some elderly healthy subjects may be due to low-grade inflammation or an undiscovered disease that influences serum YKL-40 levels. Circulating YKL-40 seems to be cleared by the kidneys, but studies are needed to determine the metabolism of circulating YKL-40, its circulating half-life and if YKL-40 is bound to substances in blood.

An automated test for determination of YKL-40 in serum will hopefully be developed in order to decrease expenses of a serum YKL-40 measurement. It is also important to develop an ELISA for determination of YKL-40 in mouse or rat for functional and pharmaceutical studies of YKL-40 in these animals.

4.1. Infectious diseases

More than 75% of patients with Streptococcus pneumoniae pneumonia (Nordenbaek et al. 1999) and Streptococcus pneumoniae bacteremia (Kronborg et al. 2002) had elevated serum concentrations of YKL-40 compared with age-matched healthy subjects ( Table 1 ). The peak in serum YKL-40 in patients with community-acquired Streptococcus pneumoniae pneumonia requiring hospitalization was seen on day 1 after hospitalization. Treatment with antibiotics of these patients resulted in decreases in serum YKL-40, reaching normal level in most patients within 7-14 days. The serum C-reactive protein (CRP) level reached normal range a few days later than serum YKL-40 (Nordenbaek et al. 1999). Patients with atypical pneumonia or Haemophilus influenzae had normal serum YKL-40 (Nordenbaek et al. 1999). YKL-40 could also be detected in the bronchioalveolar lavage fluid (BAL) from patients with tuberculosis (personal observation).

In patients with Streptococcus pneumoniae bacteremia the serum YKL-40 level was associated with the severity and fatal outcome of the disease (Kronborg et al. 2002). Serum YKL-40 was higher in patients who needed hemodialysis, pharmacological treatment of hypotension and mechanical ventilation compared to patients without the need of intensive supportive treatment. Multivariate Cox regression analysis (including serum YKL-40, cerebral symptoms, mechanical ventilation, pharmacological treatment of hypotension and hemodialysis) showed that high serum YKL-40 at time of diagnosis of Streptococcus pneumoniae bacteremia was an independent prognostic variable of poor prognosis in terms of survival from Streptococcus pneumoniae bacteremia. In the same patients serum CRP was not a prognostic marker of survival. If also the plasma concentration of soluble urokinase receptor was included in the multivariate Cox analysis serum YKL-40 was not an independent prognostic variable (Wittenhagen et al. 2004).

Østergaard et al. (2002) have shown that YKL-40 was produced locally within the compartment of an infection. Patients with purulent meningitis and encephalitis had higher YKL-40 concentrations in cerebrospinal fluid as compared with the YKL-40 levels in patients with lymphocytic meningitis and patients without meningitis. In the few patients who died of the infection the cerebrospinal concentration of YKL-40 was higher compared to the patients who survived. The overlap between cerebrospinal fluid concentrations of YKL-40 in patients with bacterial meningitis and lymphocytic meningitis was large and YKL-40 cannot be used for diagnostic purposes of patients with meningitis. Patients with non-infectious spinal diseases (i.e. cervical spondylotic myelopathy, lumbal canal stenosis and lumbar disc herniation) had higher YKL-40 concentrations in cerebrospinal fluid compared to controls or patients with scoliosis (Tsuji et al. 2002), but not as high as patients with infectious diseases.

In patients with Streptococcus pneumoniae pneumonia followed during treatment with antibiotics the changes in serum YKL-40 were parallel to that of serum lactoferrin and NGAL (proteins located in the specific granules of neutrophils like YKL-40), but not to serum MPO (a protein located in the azurophil granules), and only partial parallel to that of the total numbers of neutrophils in blood (Nordenbaek et al. 1999). In patients with Streptococcus pneumoniae bacteremia serum YKL-40 was inversely correlated with the total number of neutrophils in blood (Kronborg et al. 2002) and directly correlated with the soluble form of urokinase-type plasminogen activator receptor (expressed on different cell types including neutrophils, macrophages and lymphocytes) (Wittenhagen et al. 2004). In patients with bacterial meningitis a correlation was found between cerebrospinal fluid concentrations of YKL-40, lactoferrin and the number of neutrophils in cerebrospinal fluid and blood, but no such relationship was found in patients with lymphocytic meningitis or non-meningitis. YKL-40 is probably not released from the circulating neutrophils but is first released after the cells have reached the site of inflammation. Cerebrospinal fluid concentrations of YKL-40 from patients with bacterial meningitis, lymphocytic meningitis and non-meningitis were also correlated with the cerebrospinal fluid levels of neopterin (a protein secreted by macrophages, microgliae), indicating that YKL-40 is also secreted by activated macrophages in the cerebrospinal fluid (Østergaard et al. 2002).

Injection of healthy subjects with Esherichia coli endotoxin resulted in a significant increase in plasma YKL-40 within 2 hours and highest value is found at the 24 hours time-point after injection (Johansen et al. 2005a). The exact peak value in plasma YKL-40 was located between 8 and 32 hours after endotoxin injection, but was not further specified in the study, since blood samples were available at start and at 2, 4, 8, 24, and 32 hours after endotoxin injection. YKL-40 had a faster reaction time compared to CRP where the increase was first significant after 8 hours. Endotoxaemia is known to induce a marked increase in circulating TNF α and IL-6 already after 30 and 60 minutes with peak values at 90 minutes and 2-3 hours after endotoxin, respectively (Bundgaard et al. 2003; Krabbe et al. 2001). It is likely that regulatory relationships exist between TNF α , IL-6 and YKL-40 but the mechanisms are at present unknown.

The exact cellular source of the high serum and cerebrospinal fluid concentrations of YKL-40 in patients with infectious diseases is unknown but it probably originates from activated macrophages and neutrophils. YKL-40 is expressed by CD14+,CD16+ macrophages in patients with RA (Baeten et al. 2000) and this subpopulation of macrophages dominant often in sepsis (Fingerle et al. 1993). Macrophages, one of the most versatile cell type in the body, participate in a vast array of biological processes and are key mediators of both inflammatory functions (e.g. fighting infections) to tissue remodeling functions (e.g. wound healing) (Nathan 1987; Sunderkotter 1994). The diversity of macrophages' functional repertoire suggests that their differentiation and activation may be subject to the profound influence of environmental changes. Adherence to ECM stimulates monocytes to undergo differentiation into inflammatory or reparative macrophages and induces monocytes and macrophages to express macrophage colony-stimulating factor that promotes long-time survival, proliferation and phagocytic activities (Morrissette et al. 1999; Vaday et al. 2000). As described earlier in Chapter 1 it has been suggested that YKL-40 may be a differentiation marker of macrophages since elevated YKL-40 expression was found when monocytes differentiated to macrophages (Krause et al. 1996; Rehli et al. 1997; Renkema et al. 1998; Rehli et al. 2003), and SAGE demonstrated that YKL-40 expression was increased 288 fold after stimulation of monocytes with GM-CSF, 182 fold after stimulation with M-CSF and 31 fold in lipopolysaccharide stimulated monocytes (Hashimoto et al. 1999a; Suzuki et al. 2000). However, the role of YKL-40 in acute inflammation is unknown. Inflammatory responses to infections require the emigration of monocytes, neutrophils and T-lymphocytes from the vascular system, through endothelium, and into the ECM surrounding the injured tissue. The alveolar macrophage is poised to respond to the usual daily exposure to bacteria that enter the terminal airways and is capable of initiating an inflammatory reaction in the event that the microbial burden is too large or too virulent to be contained by the resident macrophage alone (Nelson et al. 1995). These activated alveolar macrophages can also recruit neutrophils into the alveolus from the reserves within the pulmonary microvasculature where approximately 40% of the body's neutrophils are marginated (MacNee et al. 1990; Nelson et al. 1995).

The transition of neutrophils from the vasculature into the site of inflammation elicits remarkable changes in neutrophil behavior as the cells adhere to and migrate across ECM towards inflammatory foci and the ultimate in situ elimination of foreign microorganisms through phagocytosis, generations of reactive oxygen metabolites and release of microbicidal substances. Most of the steps in this process are dependent on the mobilisation of cytoplasmatic granules and secretory vesicles (Goetzl et al. 1996; Ganz et al. 1997; Owen et al. 1999; Vaday et al. 2000; Faurschou et al. 2003). YKL-40 is a constituent of the specific granules in neutrophils (Volck et al. 1998), and proteins in these granules often participate in antimicrobial activities. However, Østergaard et al. (2002) could not demonstrate that YKL-40 had antimicrobial activity.

Conclusions and future perspectives

YKL-40 can be regarded as an acute phase protein, since its serum concentration increased by more than 25% following an inflammatory stimulus (as defined by Kushner 1982). Patients with Streptococcus pneumoniae pneumonia or bacteremia had 8-10 fold higher serum concentrations of YKL-40 compared to healthy subjects. Serum or spinal fluid concentration of YKL-40 can to some extent indicate the severity and prognosis of a bacterial infection, and serum YKL-40 might add to the information of serum CRP in patients with severe acute bacterial infections. Activated macrophages and neutrophils are probably the major source of circulating YKL-40 in infectious diseases. However, the function of YKL-40 in the inflammatory foci of a bacterial infection is unknown and will hopefully be clarified in the future.

4.2. Joint diseases

Rheumatoid arthritis

Rheumatoid arthritis (RA) is a class II-associated autoimmune disease of unknown etiology characterized by symmetrical synovial inflammation of the joints. Most patients experience a chronic fluctuating course of the disease leading to joint damage with destruction of cartilage and periarticular bone resorption. Joint deformity and disability often follow this progressive joint destruction. The mortality of RA patients with long-term disease is increased compared with that of the general population (Gabriel et al. 1999). The ultimate goal of treating RA is to induce a complete remission, but this occurs infrequently and managing RA patients are therefore to prevent joint damage and loss of function and to decrease pain.

Osteoarthritis

Secondary osteoarthritis (OA) is the most prevalent disease of articular joints and is one of the major causes of disability in the elderly (Lawrance et al. 1998). OA is part of the aging process, but its etiology is far from being fully understood. There is strong evidence that the structural changes observed in OA cartilage with appearance of fibrillations, cell clusters and changes in ECM composition and with depletion of cartilage are due to combination of several different factors like biomechanical forces and aberrant behavior of resident chondrocytes (Nuki et al. 1999; Pelletier et al. 2000; Poole 2003). Cartilage degradation and loss are the major features of OA, but the disease process also affects the synovial membrane, subchondral bone, ligaments and periarticular muscles. It is unclear which factors are responsible for the initiation of OA and which stimuli regulate the chondrocyte proliferation into cell clusters and the hyperactive phenotype of OA chondrocyte. Acute joint injury or chronic exposure of cartilage to an abnormal biochemical or biomechanical environment result in activation of chondrocytes. This chondrocyte response is manifested by enhanced cell proliferation and death, ECM degradation and new matrix synthesis, and the cells synthesize large number of proinflammatory cytokines (e.g. IL-1 β , TNF α , IL-6, IL-8, IL-11, IL-17), anti-inflammatory cytokines (e.g. IL-4, IL-10, IL-13), MMPs and their inhibitors, growth factors, matrix molecules and nitric oxide (a catabolic factor linked with chondrocyte apoptosis). Since chondrocytes are sequestered within lacunae of articular cartilage, a tissue that is avascular and aneural, it has been suggested that these cytokines act within cartilage in an autocrine or paracrine manner. IL-1 β and TNF α are of major importance to cartilage destruction and resorption of subchondral bone in OA. They stimulate their own synthesis and induce chondrocytes, synovial macrophages and fibroblasts to produce other cytokines, leukocyte inhibitory factor, proteases, prostaglandin E2 and MMPs (MacNaul et al. 1990; Brennan et al. 1992; Lotz et al. 1995; Kammermann et al. 1996; Shlopov et al. 1997, 2000). In OA patients like in RA the MMPs (particularly MMP3), TIMPs, aggrecanase, serine- and cysteine proteinases, the plasminogen activator and its inhibitors play a role in cartilage degradation (Woessner et al. 1991; Freemont et al. 1997; Shlopov et al. 1997; Pelletier et al. 2001). As proteoglycans are lost and collagen is degraded, the articular cartilage swells. Although the chondrocytes make an attempt to repair the cartilage by increasing the production of proteoglycans at some point the damage is too great and the repair response is overwhelmed.

An association exists between synovitis and progression of structural changes of the OA joints (Pelletier et al. 2001). The presence of synovial inflammation is believed to be a secondary phenomenon related to the destruction of cartilage and the release of cartilage breakdown products into the synovial fluid. In OA synovium, the inflammatory changes that take place include synovial hypertrophy and hyperplasia with increased number of lining cells and a mixed population of inflammatory cells. Some degree of synovitis is found in early stages of OA, but in patients with later stages of OA the degree of synovitis is usually mild or moderate. In severe OA the extent of inflammation and the morphological changes in the synovial membrane can be indistinguishable from that observed in RA patients (Haraoui et al. 1991; Farahat et al. 1993; Smith et al. 1997; Nakamura et al. 1999).

The most established methods to assess progression of cartilage destruction in OA patients are the measurement of joint space width using plain radiographs and determination of chondropathy by arthroscopy. MRI is more sensitive than radiography, but this method is not yet validated for monitoring patients with OA. There are no biomarkers available to diagnose OA at an early stage and no markers have so far gained acceptance in clinical routine for monitoring disease activity in OA patients. There are several biomarkers associated with cartilage degradation and synovial inflammation in OA such as serum levels of COMP, hyaluronan, the N-terminal propeptide of type IIA procollagen, MMPs, TIMPs and the urinary excretion of the C-terminal crosslinking telopeptide of type II collagen (Sharif et al. 1995; Conrozier et al. 1998; Petersson et al. 1998; Myers 1999; Garnero et al. 2000, 2001, 2002a; Pelletier et al. 2001; Poole 2002; Pavelka et al. 2004; Takahashi et al. 2004), and these markers are currently under investigation in longitudinal studies of OA patients. None of these biomarkers are specific for OA.

Ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease and is mostly characterized by inflammation of the sacroiliac joints, the enthesis, and the spine (Braun et al. 1998, 2000). Some patients also have peripheral arthritis. AS has a strong association with specific subtypes of HLA-B27 and may be a T-cell-driven disease (Sieper et al. 1995; Marti et al. 1999; Khan 2000), but its pathogenesis remain obscure and autoantigens are not completely defined. Patients with AS had YKL-40 expression in mononuclear cells in the synovial membrane (probably CD14+CD16+ macrophages) and in a subpopulation of PBMCs (Baeten et al. 2000). The number of mononuclear cells with YKL-40 expression in these patients was lower compared to RA patients. Three small studies have found elevated serum YKL-40 in patients with AS with severe disease activity and the changes in serum YKL-40 after treatment with infliximab correlated with changes in clinical outcome measures (Bath AS disease activity index) and serum CRP (D'Amore et al. 2000; Maksymowych et al. 2002; Johansen et al. 2003b).

Conclusions and future perspectives

YKL-40 is expressed by a subgroup of macrophages in the inflammed synovial membrane from patients with RA, OA and AS, and highest number of YKL-40+ cells are found in RA patients. The number of YKL-40+ macrophages in the synovial membrane of RA patients is related to the degree of synovial inflammation and joint erosion. YKL-40 is also expressed by fibroblast-like synovial cells from inflammed synovial membrane from RA patients. Furthermore, a subpopulation of articular chondrocytes located in the superficial and middle layer of arthritic cartilage from RA and OA patients express YKL-40 and the protein may reflect the heterogeneity of the OA chondrocyte phenotype. YKL-40 is found in mg levels in synovial fluid from RA and OA patients with severe synovial inflammation and is probably mainly derived from macrophages and fibroblast-like synovial cells in the inflammed synovial membrane and from activated neutrophils in the synovial fluid. A smaller amount of YKL-40 in the synovial fluid may originate from articular chondrocytes. The YKL-40 concentration in synovial fluid of RA and OA patients is related to the volume of the synovial membrane and to the serum concentration of YKL-40, indicating that a substantial amount of YKL-40 in serum from RA and OA patients originates from the arthritic joints.

The present studies suggest that serum YKL-40 reflects synovial inflammation in rheumatic joint diseases independent of etiology. Serum YKL-40 is elevated in approximately 40% of RA patients with clinically active disease and in 13% with mild or inactive disease compared to healthy subjects. Some but not all studies found elevated serum YKL-40 levels in OA patients compared to healthy subjects. Serum YKL-40 can not be regarded as a better biomarker than ESR and serum CRP to evaluate disease activity in RA patients, and a single measurement of serum YKL-40 can not predict the prognosis of the patients. Since YKL-40 is produced locally by cells in the arthritic joint, the assessment of serum YKL-40 may provide new and a more direct information of the local disease activity in arthritic joints compared to ESR and serum CRP. The relationship in RA patients between continuously elevated serum YKL-40 and the progression in bone erosions suggests that large prospective studies of RA patients should be performed to assess if the combined measurement of serum CRP and YKL-40 levels (and other biomarkers) are better to determine ongoing disease activity and to predict joint destruction than if only serum CRP is determined. It may also be of value to evaluate changes in serum YKL-40 (in combination with other biomarkers) in RA patients during treatment with TNF α , IL-1 and IL-6 blocking agents ("Biological treatments") since changes in these biomarkers may be useful to identify after a few months of treatment the patients who will respond to the treatment. The clinical value of serum YKL-40 as a potential biomarker in OA and AS patients is unclear, and longitudinal studies of these patients are necessary to assess the value of serum YKL-40 in monitoring disease activity and progression in patients with OA and AS.

YKL-40 is an autoantigen in RA and may play a pathogenic role in the inflammatory process and joint destruction of RA patients. Increased YKL-40 synthesis in the joints of RA patients may lead to an increased YKL-40-derived peptide presentation, which could amplify the local autoimmune response. It is not known if YKL-40 has a role in the development and progression of OA and AS. YKL-40 is a growth factor for fibroblast-like synovial cells, fibroblasts, chondrocytes, and endothelial cells. Locally secreted YKL-40 may have an autocrine and/or paracrine effect on synovial macrophages, fibroblast-like synovial cells, endothelial cells, and chondrocytes playing a role in their proliferation rate or protect them from undergoing apoptosis. The protein may also stimulates angiogensis in the synovial membrane, or exhibits a role in the pericellular ECM remodeling in RA, OA and AS patients.

If future studies show that YKL-40 has a function in the development and progression of RA then inhibition of YKL-40 expression or prevention of YKL-40 activity could offer an approach to suppress inflammation and prevent joint damage in RA patients. Such interventions include induction of immunological tolerance, inhibition of YKL-40 production (e.g. siRNA), and neutralization of YKL-40 activity (e.g. monoclonal antibodies against YKL-40 or its receptor, natural YKL-40 receptor antagonists). However, the effectiveness and toxicity of anti-YKL-40 based interventions are difficult to predict at present.

4.3. Diseases with chronic inflammation
and granuloma formation

Giant cell arteritis

Giant cell arteritis (GCA) is a systemic vasculitis that primarily affects medium-size and large arteries and is a T-cell-dependent disease. The etiology of GCA is unknown but may be an antigen driven disease, although the inciting antigen has not been identified. The activation of adventitial dendritic cells is an early event in the vasculitides. These dendritic cells secrete chemokines, which have a critical role in attracting T-cells and macrophages into the arterial wall. The inflammatory infiltrate in the arteries consist mainly of CD4+ T lymphocytes and macrophages that infiltrate all layers of the arterial wall. Plasma cells, neutrophils and B lymphocytes are sparse or absent in the inflammatory infiltrate. Granuloma formation is usually localized in the arterial media and multinucleated giant cells accumulate along the fragmented internal elastic lamina. The vasculitis causes arterial wall destruction with aneurysm formation and risk of rupture, proliferation of smooth muscle cells and myofibroblasts in the arterial intima with thickening of the intima that leads to occlusion of the arterial lumen and tissue ischemia. The proliferation of the intima is induced by growth factors (e.g. VEGF and PDGF) produced by giant cells and macrophages in close vicinity of the fragmented elastic laminae. Macrophages in the inflammatory infiltrates also secrete proinflammatory cytokines (e.g. IL-1 β , IL-6, TGF β ), MMPs and contribute to cellular damage through lipid peroxidation and nitric oxide synthase 2 expression. The T cells synthesize IL-2 and interferon- γ (IFN γ ). It is not known what causes the membrane to fragment initially and to subsequently initiate production of growth factors. Systemic activation of monocytes are found in GCA and it has been suggested that GCA has two components, an inflammatory reaction in vessel walls and a systemic activation of monocytes. In patients with polymyalgia rheumatica (PMR) the systemic inflammation is the major manifestation of the disease, with vascular inflammation being maintained at a subclinical level (Weyand et al. 2003a, 2003b).

It is unknown if YKL-40 is secreted by circulating monocytes from patients with GCA or PMR, but immunohistochemical analysis of temporal artery biopsies with histological signs of GCA has shown YKL-40 protein expression in multinucleated giant cells and mononuclear cells. YKL-40 expression was detected in a subset of macrophages located in areas with granulomatous inflammation at the intima-medial junction, particularly along the internal elastic membrane (Johansen et al. 1999a V). No YKL-40 expression was found in macrophages located in the intima and adventitia or in the ECM. Temporal arteries from patients with PMR have no signs of inflammation and no cells with YKL-40 protein expression. These results indicate that YKL-40 is secreted by activated macrophages at a special stage. A close relationship exists between the localization and the function of macrophages, defined on their product profile, and at least three types of macrophages can be distinguished in the lesions of GCA patients. The localization of macrophages in the blood vessel wall may be a predictor for the functional status of the macrophages, and the microenvironment of the artery may be directly involved in regulating the function of macrophages. YKL-40 expression was only found in macrophages and giant cells located in the media and intima-medial junction of the inflammed arteries. These cells have a unique product profile in that they produce MMPs, growth factors, and angiogenic factors, but not IL-1 β , IL-6 and TGF β which are produced by the macrophages homing to the adventitia (Weyand et al. 1999). Macrophages in the media and intima-medial junction are assumed to play a role in the fragmentation and destruction of the media layer and of the elastic tissue and in mediating the arterial injury response. The function of YKL-40 in GCA is unknown, but YKL-40 is an adhesion and migration factor for vascular smooth muscle cells (Nishikawa et al. 2003) suggesting that YKL-40 could have a role in the processes which lead to progression of occlusive vascular diseases like GCA.

The vascular inflammation in GCA is associated with an intense acute-phase response. High ESR is one of five components of the American College of Rheumatology 1990 criteria for the classification of GCA (Hunder et al. 1990), and ESR and serum CRP levels are used as biomarkers of disease activity in patients with GCA and PMR. However, some GCA patients have normal ESR levels at time of diagnosis and ESR provides limited information about disease reactivation in patients on a tapering regimen of prednisolone. There has been a search for new biomarkers of disease activity in patients with GCA and PMR, and at time of diagnosis patients with GCA more often had elevated plasma IL-6 than elevated ESR. Furthermore, fewer disease relapses were missed using plasma IL-6 compared to ESR, and plasma IL-6 showed prompt responsiveness to corticosteroid therapy (Weyand et al. 2000b, 2003a).

At time of diagnosis 53% of patients with GCA and 38% with PMR had elevated serum YKL-40 compared to healthy subjects (Johansen et al. 1999a V). Serum YKL-40 was significantly elevated in patients with GCA but not in PMR patients compared to controls (Table 1). During treatment of GCA patients with high doses of prednisolone serum YKL-40 decreased significantly, and after one month of treatment serum YKL-40 was normal in most GCA patients. No changes in serum YKL-40 were found in PMR patients during prednisolone treatment. 56% of the GCA patients with signs of disease relapse had corresponding elevations in serum YKL-40 (Johansen et al. 1999a V). At time of diagnosis of GCA or PMR serum YKL-40 levels correlated with ESR and serum CRP but not during prednisolone treatment. Corticosteroids are effective in suppressing clinical manifestations of GCA, but do not shorten the course of the disease or eradicate vasculitis, and only transiently down-regulate proinflammatory cytokines and have marginal effects on IFN γ (Achkar et al. 1994; Brack et al. 1997). The ability of corticosteroids to prevent blindness may result from a reduction in vascular-wall edema or the disruption of the triggering of dendritic cells in the arterial adventitia (Weyand et al. 2003b). Glucocorticoids may not have a direct effect on YKL-40 expression since some patients with GCA and PMR treated with high doses of prednisolone had unchanged serum YKL-40 despite decreases in ESR and serum CRP. The study suggests that serum YKL-40 is not a useful biomarker of disease activity in patients with PMR. Large prospective studies of patients with GCA are needed to determine if serum YKL-40 can be used as a biomarker of disease activity in GCA patients and if serum YKL-40 provides useful clinical information different from that of ESR, serum CRP and IL-6.

Inflammatory bowel disease

Inflammatory bowel disease (IBD) comprises of two forms of chronic intestinal inflammation, ulcerative colitis (UC) and Crohn's disease (CD). The etiology of IBD is unknown. It may be an autoimmune disease and mucosal immune activation is likely to play a crucial role in the pathogenesis of IBD (Sartor et al. 2004). Intestinal inflammation in IBD include a mononuclear cell and neutrophil infiltrate and mucosal ulceration with remodeling of the ECM. In both acute and chronic inflamed intestines, healing of the damaged intestinal wall requires reconstruction of the tissue framework and remodeling of ECM, and fibrosis is a nonspecific response to the chronic inflammation observed in UC and CD patients. The localization and intensity of the inflammatory cell infiltrates may be the reason for the different clinical outcomes between UC and CD. Chronic inflammation of the intestinal submucosa is characteristic of UC and an increased collagen deposition and development of fibrotic changes are largely confined to the superficial layers of the inflamed UC intestine. Whereas the inflammatory infiltrate is transmural in CD resulting in an increased transmural collagen deposition and often followed by transmural fibrosis with luminal narrowing and stricture formation (Harper et al. 1987; Matthes et al. 1992; Lawrance et al. 1999, 2001). Relapses are frequent in CD, occurring in 50% of unselected cohorts of patients per year after diagnosis (Munkholm et al. 1995). Approximately 70% of CD patients will eventually require surgery due to strictures (Sacher et al. 1990). At present it is not possible to predict relapses and ongoing fibrogenesis in IBD patients.

Neutrophils and macrophages in the inflammed intestine from patients with UC and CD had YKL-40 protein expression (Vind et al., personal communication). YKL-40 or its peptide derivatives may be a target of the T-cell-mediated immune response in IBD and could play a role in the pathogenesis of IBD. Vos et al. (2000a) found proliferative responses of PBMC from IBD patients against YKL-40 peptides (259-271 and 263-275, which were predicted to bind to DRB1*0401 with the aid of a DRB1*0401 peptide-binding motif) suggesting that YKL-40 may be an autoantigen in IBD patients and that the immune response to YKL-40 could play a role in sustaining chronic inflammation. However, no correlation was found between the disease activity score in IBD patients and the T-cell responses against the YKL-40 peptides.

Vos et al (2000b) also reported in a small study of IBD patients that some had elevated plasma YKL-40 compared to healthy controls. This has been confirmed in three larger studies of IBD patients (Koutroubakis et al. 2003; Punzi et al. 2003; Vind et al. 2003) (Table 1). 29% of patients with active UC had elevated serum YKL-40 compared to controls and their median serum YKL-40 was higher compared to the level in inactive UC patients and controls. 38% of patients with active CD had elevated serum YKL-40 compared to controls and significantly higher median serum YKL-40 compared to controls but not to inactive CD patients. Significant correlations were found between serum YKL-40 and CRP levels and Simple Clinical Colitis Activity Index in UC patients whereas low correlations were seen in CD patients between serum YKL-40, CRP and Harvey-Bradshaw score (Vind et al. 2003). In another study serum YKL-40 was elevated in active CD patients compared to inactive CD patients using the Crohn's Disease Activity Index and significant correlations were found between serum YKL-40, CRP and this disease activity score (Koutroubakis et al. 2003). IBD patients with joint involvement had higher serum YKL-40 than patients without joint involvement (Punzi et al. 2003), and serum YKL-40 was suggested as a possible biomarker of arthropathy.

The subgroup of IBD patients with elevated serum YKL-40 may not only have intestinal inflammation or arthropathy. CD patients with stenotic disease had higher serum YKL-40 than patients with non-stenotic disease (Koutroubakis et al. 2003), and serum YKL-40 in IBD patients may reflect ongoing fibrogenesis, since YKL-40 is a growth factor for fibroblasts (Recklies et al. 2002). Large prospective studies of patients with IBD are needed to evaluate if serum YKL-40 is a useful biomarker in CD patients for determining the risk of developing stricture formation.

Sarcoidosis

Sarcoidosis is a multisystem granulomatous disorder of unknown etiology characterized by the formation of noncaseating granuloma (Newman et al. 1997). Disease activity is accompanied by chronic inflammation with mononuclear cell infiltrates and granuloma formation. Even in the early stages of granuloma formation, a fibrotic response can be observed, and in some patients the fibrotic response results in substantial and irreversible organ destruction and physiologic dysfunction. Although essentially all organs of the body may be affected by sarcoidosis, the lungs are most commonly involved (Milman et al. 1990a), and some patients with chronic active pulmonary sarcoidosis succumb to respiratory failure (Milman et al. 1990b). The natural course of sarcoidosis is unpredictable in the individual patient. Many attempts have therefore been made to find biomarkers of disease activity in pulmonary sarcoidosis, which could help identify patients at risk of irreversible lung fibrosis. Angiotensin-converting enzyme (ACE) is produced by mononuclear cells and macrophages in the sarcoid granulomas (Allen et al. 1986) and determination of ACE concentrations in serum is used routinely to monitor disease activity of patients with sarcoidosis (Liberman et al. 1983; Allen 1991). However, serum ACE reflects the total granuloma mass and is not useful as a prognostic biomarker of severe irreversible organ dysfunction (Allen 1991).

Immunohistochemical analysis of lung biopsies from patients with pulmonary sarcoidosis has demonstrated that giant cells and mononuclear cells in the sarcoid lesions had YKL-40 protein expression. In a small pilot study of patients with pulmonary sarcoidosis serum YKL-40 was elevated in 63% of the patients compared to controls (Table 1), and serum YKL-40 and ACE were correlated. Patients with high serum YKL-40 had significantly lower lung diffusion capacity compared to patients with normal or slightly elevated serum YKL-40, whereas serum ACE could not discriminate between patients with low or high lung diffusion capacity (Johansen et al. 2005b). This study suggests that it needs to be evaluated in a large prospective study of patients with sarcoidosis whether the serum YKL-40 level in combination with lung function tests and other biomarkers of disease activity could be useful to monitor in order to identify at an early stage patients with sarcoidosis at high risk of irreversible lung fibrosis. It is unknown if YKL-40 is involved in the pathogenesis of sarcoidosis.

Systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune disease characterized by initial inflammation followed by fibrotic changes of the skin, blood vessels and several organs. The pathogenesis of SSc is unclear, but various growth factors are involved in the fibrotic processes (Takehara 2003). The earliest cellular changes in affected lungs from patients with SSc are lymphocyte and plasma cell infiltration of the alveolar walls and increased numbers of macrophages in the alveolar spaces (Harrison et al. 1991). BAL studies of patients with SSc show that inflammatory alveolitis is usually characterized by an increase in alveolar macrophages and neutrophils (Silver et al. 1984; Owens et al. 1986). BAL fluid neutrophilia in patients with fibrosing alveolitis is indicative of disease progression and deterioration of lung function with fibrosing alveolitis (Wells et al. 1994; Witt et al. 1999). Interstitial fibrosis, vascular lesions including fibrous thickening of the intima, media hypertrophy, and perivascular fibrosis are found in SSc lungs and lead to a restrictive lung function pattern and impairment of the diffusing capacity (Weaver et al. 1968; Harrison et al. 1991).

Immunohistochemical analysis of a lung biopsy from a SSc patient with lung fibrosis showed YKL-40 protein expression in mononuclear cells in areas with inflammation but not in fibroblasts or in the ECM (Nordenbæk et al. 2005). Serum YKL-40 was elevated in 27%-35% of patients with SSc compared to controls (Table 1). Serum YKL-40 correlated with serum levels of soluble IL-2 receptor α and the procollagens PIIINP and PINP. Serum YKL-40 was significantly elevated in patient with arthritis, pulmonary fibrosis by chest X-ray, obstructive ventilatory pattern, reduced lung diffusing capacity and with skin retraction compared to patients without these findings. Furthermore SSc patients with elevated serum YKL-40 had shorter survival and died more often due to extensive interstitial or vascular fibrosing processes (e.g. pulmonary fibrosis, SSc renal crisis) than patients with normal serum YKL-40 (Montagna et al. 2003; Nordenbæk et al. 2005). It is unknown if YKL-40 plays a role in the pathogenesis of pulmonary fibrosis in patients with SSc. YKL-40 is a growth factor of fibroblasts (Recklies et al. 2002) and it is possible that YKL-40 takes part in the ECM remodelling process and development of fibrosis in patients with SSc. Large prospective studies of patients with SSc are needed to determine if patients with high serum YKL-40 have a poor prognosis and are at risk of developing severe organ fibrosis.

Heart transplantation

Cellular rejection is a major problem for heart, lung and kidney transplant patients. Biopsies are used for detecting transplant rejection, however the procedures are associated with morbidity and mortality. Noninvasive methods to determine transplant rejection are needed. A small pilot study of 25 heart transplant recipients found that serum YKL-40 was significantly higher in these patients compared to controls and related with the number of years since transplantation and of moderate- to high-grade rejection episodes (Fiore et al. 2000). Studies are needed to evaluate if macrophages in areas of transplant rejection express YKL-40 and if serum YKL-40 can be a biomarker in the follow-up of patients with organ transplants.

Conclusions and future perspectives

YKL-40 is produced by macrophages, giant cells and neutrophils in areas with inflammation in affected tissues from patients with diseases characterized by chronic inflammation and development of fibrosis as illustrated in patients with GCA, IBD, sarcoidosis and SSc. It has to be evaluated if YKL-40 is involved in the pathogenesis of these diseases. Serum YKL-40 is not useful as a biomarker for diagnosis of GCA, IBD, sarcoidosis and SSc, but the studies indicate that serum YKL-40 may be a valuable biomarker for monitoring disease activity in these patients. Relationships are found between serum YKL-40 and CRP and clinical parameters of disease activity in patients with chronic inflammation, but serum YKL-40 reflects other aspects of the inflammatory response than serum CRP does. YKL-40 is produced locally in tissue with inflammation, unlike CRP production by hepatocytes in the liver. Serum YKL-40 and CRP levels may complement each other in the determination of disease activity in patients with chronic inflammation. YKL-40 stimulates growth of fibroblasts in vitro but the exact biological functions of YKL-40 in diseases with chronic inflammation, granuloma formations and development of fibrosis are unknown. Elevated serum YKL-40 in patients with IBD, sarcoidosis and SSc may reflect ongoing fibrogenesis that may lead, after a variable time, to clinical manifestations of severe organ fibrosis, like symptoms of fibrostenosis in the intestine of patients with CD and affected lung function in patients with sarcoidosis. Prospective longitudinal studies of changes in serum YKL-40 in these patients are needed to evaluate if serum YKL-40 can be used as a biomarker of ongoing fibrosis and to predict disease relapse.

4.4. Liver fibrosis

Liver fibrogenesis represents the wound healing response of the liver and is a dynamic process with phases of either net matrix deposition or net matrix degradation leading respectively to progression or regression of liver fibrosis. The process leading to liver fibrosis includes three phases following liver tissue injury: acute inflammation, synthesis of collagenous and non-collagenous ECM components, and tissue remodeling (Bedossa et al. 2003). The ECM components of the fibrotic matrix are similar to those present in the normal liver, but in the fibrotic liver all ECM components are increased in quantity. ECM of the normal liver constitutes approximately 0.5% of the liver wet weight whereas the cirrhotic liver contains approximately six times more ECM than the normal liver. In normal liver the interstitial ECM is found in the portal area, around the central veins and in the liver capsule. The subendothelial space of Disse separates the hepatocytes from the sinusoidal endothelium and contains both an interstitial and a basement membrane-like ECM of low density. In the fibrotic liver collagenous components, particularly collagen types I and III, increase up to tenfold predominantly in the periportal and perisinusoidal space, and the perisinusoidal low-density ECM is transformed to a high-density ECM. The 3 cell types delimiting the space of Disse, hepatocytes, hepatic stellate cells (HSC, also known as Ito cells, lipocytes or fat-storing cells) and endothelial cells, all express ECM components. HSCs play a cardinal role in the pathogenesis of liver fibrosis and are the major producers of the fibrotic neomatrix and control ECM turnover (Friedman 1993, 1999; Li et al. 1999; Schuppan et al. 2001; Bedossa et al. 2003). HSCs are thought to be derived from mesenchymal cells. In the normal liver quiescent HSCs represent 5-8% of the total number of liver cells and are the major storage sites of vitamin A and are important sources of paracrine, autocrine, juxtacrine and chemoattractant factors that maintain homeostasis in the micro-environment of the hepatic sinusoid (Geerts 2001).

Following chronic liver injury (e.g. viral hepatitis infection, alcoholic or drug toxicity or any other factor that cause damage to hepatocytes) an inflammatory reaction in the liver is elicited. The damaged hepatocytes, their membrane components, metabolites of toxic agents, and infiltrating inflammatory cells activate the Kupffer cells, which release cytokines and growth factors (e.g. TGF β , PDGF, TNF α ) and reactive oxygen species. These factors (particularly TGF β ) stimulate HSCs, which then proliferate, differentiate, and undergo a major phenotypic transformation to a highly proliferative "myofibroblast-like" phenotype. Platelets and infiltrating leukocytes are also sources of cytokines that participate in HSC activation. The activated HSCs secrete a large amount of ECM components (e.g. type III collagen, structural glycoproteins, proteoglycans, hyaluronan), MMPs, TIMPs, and growth factors (e.g. VEGF, IGF-1, TGF β , hepatocyte-, epidermal-, acidic fibroblast-, connective tissue- and hematopoietic growth factors) (Olaso et al. 1998; Maher et al. 1999; Friedman et al. 2000; Pinzani et al. 2000; Oh et al. 2001). An increase in the production of TIMPs occurs in the early stages after liver injury and persists throughout the time course of development of liver fibrosis. Due to relatively less MMPs production and an increased synthesis of TIMPs the overall net consequence leads to an enhanced deposition of ECM (Benyon et al. 2001; Schuppan et al. 2001). If the source of liver injury is removed a single liver tissue injury most often results in an almost complete resolution. During the recovery phase there is an increased MMP activity in the liver, a decrease in the expression of TIMPs, and increased apoptosis of activated HSCs. Regression of liver fibrosis is therefore characterized by degradation of fibrillar liver matrix and restoration of normal liver histology (Iredale et al. 1998; Iredale et al. 2001). Conversely, the persistence of the original liver toxic agent causes the prolonged activation of the tissue repair mechanisms, thus leading to liver fibrosis rather than to effective liver repair.

A liver biopsy is the key examination for the diagnosis and staging of liver fibrosis. However, the use of a liver biopsy can not be used as a general screening procedure for liver fibrosis or used repeatedly to monitor disease progression during the follow-up of patients with liver fibrosis. A liver biopsy has some discomfort for the patient, it requires hospitalization, and there is a risk of complications with a mortality of 0.015% (Piccinino et al. 1986). There is also a sampling error of approximately 24% false-negative (Nord 1982) and the liver biopsy does not reflect the dynamic of fibrous tissue turnover. Noninvasive methods to determine liver fibrosis are therefore needed both to diagnose significant liver fibrosis and to monitor effects of therapy on fibrogenesis and fibrolysis. Consequently there has for many years been a search for biomarkers of liver fibrosis. Several such markers have been proposed (e.g. hyaluronan, PIIINP, MMP-2, MMP-9 and TIMP-1) (Bentsen 1992; Oh et al. 2001), but none are yet used routinely in the clinical practice. However, it has been forested that a panel of biomarkers of liver fibrosis in the future will replace sequential liver biopsy as a standard care and that serum YKL-40 may be one of these markers (Oh et al. 2001).

YKL-40 expression in the liver

In 1993 Hakala et al. reported that YKL-40 mRNA was strongly expressed in human liver tissue, but it could not be determined whether the mRNA used in the experiments originated from normal or fibrotic liver tissue. Hu et al. (1996) was unable to demonstrate YKL-40 mRNA expression in normal liver tissue. Immunohistochemical analysis of liver biopsies from patients with different liver diseases have shown YKL-40 protein expression in areas with slight fibrosis (either pericellular or perisinusoidal), along fibrotic septa in association with signs of fibrogenesis, and in areas with moderate and severe fibrosis (Johansen et al. 1997, 2000a VI). Hepatocytes did not express YKL-40 protein and no expression was found in normal liver tissue except in mesenchymal structures of the portal tract. Patients with chronic active HCV had YKL-40 protein expression in areas with piecemeal necrosis but not in the lymphocytes. YKL-40 protein expression was found in 90% of liver biopsies with moderate or severe fibrosis, in 86% with slight fibrosis, and in 93% with fibrogenesis. YKL-40 expression was found in ECM both in areas free of cells as well as in cellular areas (Johansen et al. 2000a VI). It was not possible by the immunohistochemical method to discriminate the extent to which the YKL-40 protein expression was intracellular, and if HSCs, leukocytes and macrophages expressed YKL-40. These 3 cell types are probably responsible for YKL-40 synthesis in the fibrotic liver, and ongoing in vitro studies demonstrate that HSCs express YKL-40 mRNA (Eva Efsen, unpublished). YKL-40 is a growth factor for fibroblasts, works synergistically with IGF-1 (Recklies et al. 2002), and YKL-40 may play a role in the pathological conditions leading to liver fibrosis. Recently Shackel et al. (2003) showed using suppression subtractive hybridization and quantitative real-time RT-PCR that YKL-40 was one of the most differentially expressed genes in liver tissue from endstage cirrhosis due to HCV compared to non-diseased liver tissue, primary biliary cirrhosis and autoimmune hepatitis associated cirrhosis.

Serum concentrations of YKL-40 in patients with liver disease
in relation to liver fibrosis, disease activity and prognosis

Haemodynamic investigations with catherization of the liver vein and femoral vein have shown that YKL-40 was released from the hepatosplanchnic area (Johansen et al. 1997). The serum concentration of YKL-40 in the hepatic vein was significantly higher than in the femoral artery both in patients with liver diseases and in subjects with normal liver function. Furthermore, the hepatic venous-arterial difference in serum YKL-40 concentration and the release rate of YKL-40 from the hepatosplanchnic area were higher in patients with liver disease compared to subjects with normal liver function.

Six studies have evaluated the serum concentrations of YKL-40 in patients with liver diseases. All found elevated serum YKL-40 in patients with liver fibrosis (defined from a liver biopsy) compared to healthy controls (Johansen et al. 1997, 2000a VI; Nunes et al. 1998; Tran et al. 2000; Nøjgaard et al. 2003a, 2003b). The highest serum YKL-40 levels were found in patients with alcoholic cirrhosis and in patients with cirrhosis caused by chronic hepatitis C or B virus (Table 1), indicating that elevations in serum YKL-40 in patients with liver fibrosis are independent of disease etiology. Figure 3 illustrates the individual serum YKL-40 levels in patients with different liver diseases according to the degree of liver fibrosis. Highest serum YKL-40 were measured in patients with moderate and severe liver fibrosis (Johansen et al. 2000a VI). Nøjgaard et al. (2003a) demonstrated in a large group of patients with alcoholic liver disease that serum YKL-40 was useful to discriminate between the absence and presence of fibrosis but could not discriminate between the different degrees of liver fibrosis. 75% of the alcoholics with moderate or severe fibrosis had elevated serum YKL-40 compared to healthy controls whereas only 26% of the alcoholics without liver fibrosis had elevated serum YKL-40. Serum PIIINP was found useful to discriminate between the different degree of liver fibrosis. Serum YKL-40 was also increased in the presence of liver inflammation (Tran et al. 2000), and patients with liver fibrosis in combination with alcoholic hepatitis had higher serum YKL-40 compared to patients with fibrosis but without alcoholic hepatitis (Johansen et al. 2000a VI; Nøjgaard et al. 2003a). This suggests that some of the circulating YKL-40 in patients with alcoholic liver disease originate from activated neutrophils, which play a role in the pathophysiology of alcoholic hepatitis. The morphological features in alcoholic hepatitis include liver-cell damage, inflammatory cell infiltrate of predominantly neutrophils, and fibrosis (Poulsen et al. 1979; Baptista et al. 1988). Neutrophilia is frequent and the neutrophils are activated in alcoholic hepatitis and produce proinflammatory cytokines, chemokines and reactive oxygen species (Taieb et al. 2000). The role of YKL-40 secreted from the activated neutrophils in areas with alcoholic hepatitis is unknown, but it may participate in maintaining the liver inflammation, activate the HSCs and stimulate ECM production.

Serum concentrations of YKL-40 in patients with liver diseases correlated with other ECM products secreted by HSCs (serum PIIINP, hyaluronan, MMP-2 and TIMP-1). Low correlations were found between serum YKL-40 and enzymes secreted by hepatocytes (serum aspartate aminotransferase and alkaline phosphatase) and inverse correlations were found with serum albumin and the coagulation factors 2,7 and 10 (Johansen et al. 1997, 2000a VI; Tran et al. 2000; Nøjgaard et al. 2003a, 2003b). Serum YKL-40 correlated with other parameters reflecting the degree of liver fibrosis, such as hepatic venous pressure gradient and the postsinusoidal resistance and inversely with the clearance of indocyanine green (Johansen et al. 1997).

Nunes et al. (1998) found that serum YKL-40 decreased in patients with chronic HCV who responded to interferon treatment and Nøjgaard et al. (2003b) found that patients with chronic HCV treated for 12 months with alpha-interferon and ribavirin had a decrease in serum YKL-40 at 6 months after the end of treatment. In the patients who responded to treatment, serum YKL-40 was not related to changes in HCV titer or the liver enzymes during 12 months of treatment. The serum YKL-40 level before therapy could not predict whether a patient would respond to treatment, but the non-responders had unchanged high serum YKL-40 during the 12 months treatment period and at 6 months after therapy.

Nøjgaard et al. (2003a) reported that patients with alcohol induced liver disease and high serum YKL-40 had shorter survival than alcoholics with normal serum YKL-40 (Relative risk = 4.24, 95% confidence interval 2.18-8.26, p < 0.0001). Multivariate Cox regression analysis including serum YKL-40 and variables known to have prognostic information of survival in alcoholics (i.e. years of high alcohol intake, serum creatinine, coagulation factors 2,7, and 10, alkaline phosphatase and IgM) showed that serum YKL-40 had no independent prognostic value.

Conclusions and future perspectives

Increased YKL-40 mRNA and protein expressions are found in fibrotic liver tissue from patients with alcoholic liver disease and chronic HCV infection. Serum concentrations of YKL-40 are elevated in most patients with moderate to severe liver fibrosis and cirrhosis, independently of disease etiology, and may provide new information of ongoing fibrogenesis in the liver. Patients with alcoholic liver disease and high serum YKL-40 have a poorer prognosis compared to patients with normal serum YKL-40. Large prospective studies of patients with liver diseases are needed to determine if patients with slight liver fibrosis and high serum YKL-40 are at risk of developing cirrhosis, and if serum YKL-40 in combination with other biomarkers of liver fibrosis (e.g. serum hyaluronan and PIIINP) can predict the severity of liver fibrosis and be used in monitoring patients with liver fibrosis or cirrhosis. Serum YKL-40 may also be useful to monitor in patients with liver diseases during anti-fibrotic or anti-viral therapy. The biological function of YKL-40 in liver diseases is not known and it needs to be determined if YKL-40 has a role in the pathogenesis of liver cirrhosis. As has been found for fibroblasts YKL-40 may be a growth factor for HSCs and could stimulate their production of collagen. Reducing the ECM production by activated HSCs is crucial in preventing liver fibrosis. If YKL-40 has a role in development of liver fibrosis then inhibition of YKL-40 production or blocking of YKL-40 activity in patients with alcoholic liver disease or hepatitis C or B virus may be a valuable method to inhibit the development of liver fibrosis.

Is serum YKL-40 a new biomarker
in cancer patients?

Acceptance of novel tumor markers in clinical settings requires thorough validation before being implemented into routine clinical use. Werner et al. (1993) have suggested that "Tumor markers" are classified according to six different clinical criteria such as biochemical characteristics, organ specificity or clinical usefulness in order to assess the value of tumor markers in clinical practice:

1. "The marker is produced exclusively by specific tumor cells (tumor specific)"?

YKL-40 is not specific for cancer or a certain type of tumors. YKL-40 is produced by non-malignant cells (as described in Chapter 1 and 4) and by cancer cells of widely different types of solid cancer.

2. "The marker is absent in healthy or benign disease (high specificity)"?

Serum concentrations of YKL-40 do not have high specificity for cancer. YKL-40 is detected in serum from healthy subjects and elevated serum YKL-40 (compared to healthy subjects) are found in patients with non-malignant diseases such as severe bacterial infections, active RA, GCA, IBD, lung sarcoidosis, and liver fibrosis (as described in Chapter 4). Most patients with these diseases will have some clinical symptoms of their disease.

3. "The marker is present frequently in the targeted malignancy (high sensitivity)"?

In 1995 Johansen et al. (II) reported that some patients with metastatic breast cancer had increased serum YKL-40 compared to healthy subjects, and that the highest serum YKL-40 were found in patients with short survival ( Figure 4 ). This study suggested that serum YKL-40 might be useful as a prognostic marker in breast cancer patients. Recent studies have found elevated serum YKL-40 in a subgroup of patients with seven different types of localized or metastatic solid cancer compared to healthy subjects ( Table 3 ) (Cintin et al. 1999, 2002; Tanwar et al. 2002; Dehn et al. 2003; Geertsen et al. 2003; Høgdall et al. 2003; Jensen et al. 2003; Johansen et al. 2003a, 2004; Dupont et al. 2004; Brasso et al. 2006). It needs to be determined if YKL-40 is elevated in serum of patients with hematological malignancies.

Preoperative serum YKL-40 levels were elevated in 19% of patients with primary breast cancer and the patients with metastases to axillary lymph nodes had higher serum YKL-40 compared to lymph node negative patients (Johansen et al. 2003a). In patients with first recurrence of breast cancer serum YKL-40 was elevated in 31-41% of the patients, and high serum YKL-40 was associated with metastatic sites and large tumor load: 9-20% of patients with recurrence to lymph nodes or skin only had elevated serum YKL-40, 24-35% with bone metastases, and 57-61% with visceral metastases (Johansen et al. 1995; Jensen et al. 2003). Highest serum YKL-40 were found in patients with more than two different metastatic sites (Jensen et al. 2003). Preoperative serum levels of YKL-40 from patients with colorectal cancer was elevated in 26% and there was an association between serum YKL-40 and Dukes' stage: 16% of the patients with Dukes' A (tumor confined within the bowel wall, no lymph-node metastases), 26% with Dukes' B (tumor extending through the bowel wall, no lymph-node metastases), 19% with Dukes' C (regional lymph-node metastases), and 39% with Dukes' D disseminated disease) had elevated preoperative serum YKL-40 (Cintin et al. 1999). Serum YKL-40 decreased significantly after curative operation for colorectal cancer in patients with high preoperative serum YKL-40 (Cintin et al. 2002), indicating that serum YKL-40 reflect tumor burden. Preoperative serum YKL-40 was elevated in 65% of stage I and II ovarian cancer patients (Dupont et al. 2004), in 74-91% of patients with ovarian cancer stage III (tumor growth involving one or both ovaries with wide-spread intraperitoneal metastases) and IV (disseminated disease) (Høgdall et al. 2003; Dupont et al. 2004) and in 55% of ovarian cancer patients at time of first recurrence (Dehn et al. 2003). In patients with small cell lung cancer 22% with local disease and 40% with extended disease had elevated serum YKL-40 (Johansen et al. 2004). 43% of patients with metastatic prostate cancer (Brasso et al. 2006) and 83% of patients with metastatic renal cell cancer (Geertsen et al. 2003) had elevated serum YKL-40. In patients with glioblastoma serum YKL-40 was related to tumor grade and burden: 72% of patients with glioblastoma multiforme and 57% with lower grade gliomas had high serum YKL-40 (Tanwar et al. 2002).

These studies demonstrate that serum YKL-40 does not have a high sensitivity for solid carcinoma, suggesting that not all tumors express YKL-40 or it is secreted at a low level. This could be evaluated in immunohistochemical and in situ hybridization studies of biopsies from different types of cancer.

4. "The marker is detectable in early stage subclinical disease (useful for screening)"?

Determination of serum YKL-40 concentrations cannot be used as a single screening test for cancer. At time of first cancer diagnosis 16-74% of the patients had elevated serum YKL-40, and only 16-26% of patients with primary localized cancer had elevated serum YKL-40. However, in patients with locally advanced or metastatic cancer at the time of diagnosis serum YKL-40 levels were elevated in 39-83%. A high serum YKL-40 in a subject without any known disease may therefore indicate non-symptomatic cancer. Serum concentrations of YKL-40 were independent of serum CEA in colorectal cancer patients (Cintin et al. 1999, 2002), of serum CA-125 in ovarian cancer patients (Dehn et al. 2003; Høgdall et al. 2003), of serum HER-2 in metastatic breast cancer patients (Jensen et al. 2003), of serum LDH in patients with small cell lung cancer (Johansen et al. 2004) and of serum PSA in patients with metastatic prostate cancer (Brasso et al. 2006). The studies indicate that serum YKL-40 reflects other aspects of tumor growth and metastasis than these tumor markers. It may be of value to include serum YKL-40 as a biomarker for screening of cancer together with a panel of other tumor markers and imaging techniques, since an elevated serum YKL-40 level seems to reflect metastatic disease and secretion from a subset of tumors with a more aggressive phenotype and with a poor prognosis (described below).

5. "The marker's concentration reflects prognosis for an individual patient (prognosticator)"?

Eight studies have demonstrated that elevated serum YKL-40 in patients with breast-, colorectal-, ovarian-, kidney-, small cell lung-, and prostate carcinomas was an independent prognostic parameter of short recurrence free interval and short overall survival with hazard ratios between 1.3 and 4.1 ( Table 4 ). This observation was found in patients with local or metastatic cancer, and at the time of first cancer diagnosis or at the time of relapse (Johansen et al. 1995, 2003a, 2004; Cintin et al. 1999, 2002; Dehn et al. 2003; Geertsen et al. 2003; Høgdall et al. 2003; Jensen et al. 2003; Brasso et al. 2006).

High preoperative serum YKL-40 level in patients with primary breast cancer was an independent prognostic parameter of short recurrence free interval and short overall survival when axillary lymph node- and estrogen receptor status, age, tumor size and histology, menopausal status and serum YKL-40 were included in the multivariate Cox analysis (Johansen et al. 2003a). There are no longitudinal studies of the changes in serum YKL-40 levels in breast cancer patients after operation and adjuvant chemo-, antiestrogen- or radio-therapy. However, an elevated serum YKL-40 in breast cancer patients at time of first recurrence predicted shorter time to progression and shorter overall survival (Jensen et al. 2003). Multivariate Cox analysis (including estrogen receptor- and axillary lymph node status at primary diagnosis, liver metastases, more than two metastatic sites, symptomatic disease at recurrence and serum HER-2 and YKL-40 levels) showed that high serum levels of YKL-40 and HER-2 were independent prognostic variables of short time to disease progression and death (Jensen et al. 2003). Figure 5 illustrates survival curves in patients with metastatic breast cancer according to elevated or normal serum concentrations of YKL-40 and HER-2 at time of first relapse (Jensen et al. 2003). Patients with both high serum YKL-40 and HER-2 level had the poorest median survival of only 9 months contrasting 32 months for patients with normal serum YKL-40 and HER-2.

High preoperative serum concentration of YKL-40 in patients with colorectal cancer was also an independent prognostic parameter of short recurrence free interval and short overall survival (the multivariate Cox analysis included Dukes' stage, age, gender, serum CEA and YKL-40) (Cintin et al. 1999). In stage III ovarian cancer patients a high preoperative serum YKL-40 was an independent prognostic parameter of short survival (the multivariate Cox analysis included serum YKL-40 and CA-125, optimal vs. suboptimal results from primary surgery, age, and histological type of tumor) (Høgdall et al. 2003) and a similar result was found in patients with recurrence of ovarian cancer (the multivariate Cox analysis included serum YKL-40 and CA-125, age, localization of tumor and its size, performance status, primary and second-line treatment) (Dehn et al. 2003). An elevated serum YKL-40 was also an independent prognostic variable of short survival in patients with metastatic prostate cancer (the multivariate Cox analysis included age, performance status, tumor grade, serum PSA, total and bone alkaline phosphatase, PINP, crosslaps and YKL-40) (Brasso et al. 2006), and in patients with metastatic renal cell carcinoma (the multivariate Cox analysis included serum YKL-40, performance status, number of organ disease sites, organ site involvement, prior nephrectomy, and time from diagnosis to metastases) (Geertsen et al. 2003). In patients with small cell lung cancer a high serum YKL-40 at time of diagnosis and before chemotherapy was a parameter for death within the following 6 months and was independent of age, sex, disease stage, performance status, and serum LDH (Johansen et al. 2004).

In all six different types of carcinoma tested a high serum YKL-40 level was related to poor prognosis, and serum YKL-40 was independent of other known prognosticators when tested in multivariate Cox analysis. These results suggest that serum YKL-40 may be a useful "prognosticator" identifying a subgroup of cancer patients with a poor prognosis. The function of YKL-40 in cancer diseases is unknown, but these clinical studies indicate that the elevated serum YKL-40 level found in some cancer patients reflects YKL-40 secretion from a subset of tumors with a more aggressive phenotype and a poor prognosis. It is of major importance to evaluate if YKL-40 has a role in promoting growth, invasion and metastasis potential of the cancer cells.

"The marker's degree of expression correlates with therapeutic results (useful for monitoring)"?

One study of curatively operated colorectal cancer patients has evaluated changes in serum YKL-40 levels during the follow-up after surgery (Cintin et al. 2002). It was found that patients with elevated serum YKL-40 six months after the operation had significantly shorter recurrence free interval and overall survival than patients with normal serum YKL-40 at 6 months postoperative. This result was independent of serum CEA levels at 6 months postoperative. Multivariate Cox analysis scoring serum YKL-40 as a time-dependent covariant and including age, Dukes' stage, gender, and tumor localization showed that a high serum YKL-40 postoperatively in curatively operated colorectal cancer patients increased the risk of recurrence within the following 6 months by 6.9 fold and the risk of death by 8.5 fold.

The result of this study indicates that serum concentrations of YKL-40 may be useful for monitoring of cancer patients. However, large longitudinal studies of patients with other types of cancer are needed to evaluate if determination of serum YKL-40 in combination with other prognostic tumor biomarkers can be useful to monitor in cancer patients after primary operation, adjuvant chemo-, antihormonal-, and radio-therapy in order to detect first recurrence early. It is unknown if pretreatment serum YKL-40 levels or early changes during treatment can help to identify patients who respond or do not respond to the given therapy. Longitudinal studies are also needed to evaluate if serum YKL-40 can provide clinical information about disease progression in patients with metastatic cancer before this is detected by routine methods. The present studies show elevated serum YKL-40 particularly in patients with metastases in the liver and lung.

YKL-40 and liver metastases

Liver metastases imply a poor prognosis and often develop in patients with breast-, colorectal-, ovarian-, and lung- carcinoma after removal of the primary tumor. High serum YKL-40 levels are often found in patients with liver metastases (Johansen et al. 1995; Cintin et al. 1999, 2002; Dehn et al. 2003; Jensen et al. 2003). The development of liver metastases involves several steps, i.e. exfoliation of cancer cells from the primary site, entry into the portal system, adhesion to the endothelium and subsequent extravasation in the hepatic microvasculature, and multiplication and formation of glandular or acinar structure in the liver parenchyma. Whether or not cancer cells successfully metastasize to the liver depends not only on their cytological properties but also on the hepatic microenvironment involving macrophages and natural killer cells, which constitute the defense system of the liver. Myofibroblasts are usually associated with cancers of epithelial origin and contribute to the growth of metastatic tumors before neovascularization is induced (Schmitt-Graff et al. 1994). Hepatic stellate cells (HSCs) are the only mesenchymal cells present in the extravascular space of the liver parenchyma. While quiescent in the steady state, they are activated by various stimuli and undergo transformation into myofibroblasts (see also Chapter 4.4). HSCs have been shown to be activated and accumulated around the tumor cells in liver metastases from human colon carcinoma, but the complete interaction between cancer cells and HSC is not well understood. Cancer cells that metastasize to the liver release HSC activating factors and may contribute to the progression of hepatic metastasis. Furthermore growth factors (e.g. PDGF and hepatocyte growth factor) secreted by HSCs augment proliferation and migration of cancer cells in vitro (Olaso et al. 1997; Shimizu et al. 2000; Lunevicius et al. 2001). Hepatic myofibroblasts promote the proliferation of hepatocellular carcinoma cell lines, and the latter cells in turn activate and promote proliferation of the former cells (Neaud et al. 1997; Faouzi et al. 1999) demonstrating bi-directional interactions between cancer cells and HSCs. YKL-40 stimulates fibroblasts in vitro (Recklies et al. 2002) and one could speculate that YKL-40 secreted by metastatic tumor cells in the liver has an effect on HSCs and myofibroblasts, and that YKL-40 secreted from HSCs activates the cancer cells.

Conclusions and future perspectives

In order to propose guidelines on how promising tumor markers progress from the laboratory into the clinic, Hayes and colleagues have introduced the "Tumor Marker Utility Grading System" (TMUGS) (Hayes et al. 1996, 1998). According to this system, serum YKL-40 is on the "Utility scale +" or "Utility scale +/-". YKL-40 is neither organ nor tumor specific, but the present eleven retrospective clinical studies of 1605 patients with different types of cancer indicate that serum concentrations of YKL-40 may be useful as a "prognosticator" and may also have a role in screening and monitoring of cancer patients. Elevated serum concentrations of YKL-40 were found in a subgroup of patients with seven different types of solid carcinoma (including several types of adenocarcinomas, small cell carcinoma, and glioblastoma). The highest serum YKL-40 levels were found in patients with metastatic cancer and with the poorest prognosis and serum YKL-40 provided independent information of survival. The potential values of serum YKL-40 as a biomarker in monitoring and diagnosis of cancer need more studies, and its role as a biomarker in hematological malignancies has to be determined.

According to the "TMUGS", a number of validation requirements are suggested which have to be fulfilled before the marker can be considered to have reached level of evidence I ("LOE I"), whereupon clinical implementation is feasible. Most tumor marker studies are "LOE III", defined as retrospective studies where samples are not originally collected with the intent of testing the value (e.g. prognostic value) of the marker of interest. The intermediate level "LOE II" is constituted by companion studies with prospectively collected specimens as part of a therapeutic trial with pre-established endpoints and evaluation of both the marker and the therapeutic intervention. Finally "LOE I" studies are either 1) highly-powered prospective studies specifically addressing the issue of the utility of the marker or 2) an overview or meta-analysis of studies, each of which have a lower level of evidence.

It is yet unknown if knowledge of the serum YKL-40 level in an individual patient can be reliably used to make clinical decisions that will improve the outcome of the patient. All the present studies of cancer patients regarding serum YKL-40 as a tumor marker in cancer patients are retrospective in design and include a fairly small number of patients and are of a lower level of evidence "LOE III". There are therefore limitations to the conclusions that can be made from the present studies. The prognostic value of serum YKL-40 levels in patients with different types of primary and advanced carcinoma should be confirmed in additional large retrospective studies from other research groups. If a sufficient number of large retrospective studies of high quality confirm the association between serum YKL-40 and poor prognosis a summarising meta-analysis can bring the generated data to "LOE I". According to the "TMUGS" guidelines, the next step would be to launch an appropriate prospective study where the benefit of using serum YKL-40 levels in the clinical decision-making process is assessed. Endpoints should include overall survival, disease-free survival, quality of life and cost-effectiveness. The study could be designed either as a single, highly-powered, prospective, controlled study with the primary objective of testing serum YKL-40 level as a "prognosticator" or a similar prospective study where the primary goal could be the testing of a therapeutic hypothesis and secondly testing serum YKL-40 as a biomarker.

A major issue to explore is the question: "Can YKL-40 or its receptor(s) be potential targets for cancer therapy?". Unfortunately, the biological function of YKL-40 in cancer development and metastases is unknown and the elucidation of a possible function of YKL-40 in cancer diseases is an important objective of future studies. It has been shown that YKL-40 exhibits growth factor activity for cell types involved in tissue remodeling processes, and it has been suggested that YKL-40 has a role in cancer cell growth and survival, the inflammatory process around the tumor, angiogenesis, and remodeling of the ECM surrounding the cancer cells as pointed out previously. Based on the present clinical studies of serum YKL-40 levels in cancer patients one could hypothesize that YKL-40 will prove to have a role in the ability of cancer cells to proliferate, survive, invade and metastasize and/or a regulating role in cancer cell-matrix interactions and in the production of the altered extracellular matrix surrounding the cancer cells. Provided future studies show that YKL-40 has such roles, YKL-40 could be an attractive target in the design of anticancer therapy. Any approach that would inhibit the function of YKL-40 (e.g. inhibition of YKL-40 gene expression, protein synthesis and secretion, neutralization of YKL-40 activity, blocking YKL-40 conversion from a latent to an active form, interruption of YKL-40 affinity or reaction with its receptor) may limit cancer growth and metastases and improve the survival of cancer patients with YKL-40 expressing tumor cells. Potential inhibitors of YKL-40 activity include methods to inhibit YKL-40 production (e.g. siRNA), human (or humanized) monoclonal antibodies specific for YKL-40 or its receptor(s), YKL-40 receptor antagonists, or substrate molecules that competitively bind to YKL-40. Such potential inhibitors of YKL-40 could be expected to have therapeutic efficacy in cancer patients with tumors that produce YKL-40. It is therefore of major importance to explore if YKL-40 could become a target for the development of new cancer therapeutics.