Several other receptors and co-receptors have been identified to be expressed on the surface of NK cells such as DNAX accessory molecule-1 (DNAM- 1), CD69, and 2B4. DNAM-1 is a transmembrane glycoprotein with two immunoglobulin-like domains expressed on the surface NK and T cells in association with lymphocyte function antigen-1 (LFA-1) (Shibuya et al, 1996). It is a signal transducing adhesion molecule that is involved in the adhesion of certain tumour cells to CTLs and NK cells and mediates their cytotoxicity (Shibuya et al, 1996; Lakshmikanth et al, 2009). Anti-DNAM-1 mAbs inhibits T and NK cell-mediated cytotoxicity against a variety of tumour cell targets and blocks cytokine production by alloantigen-specific T cells (Shibuya et al, 1996). Recent studies also demonstrated
42 that DNAM-1 can synergize with other activating NK cell receptors in the pro- inflammatory cytokine secretion by these cells (Fauriat et al, 2010).
CD69, also known as activation inducer molecule or early T- cell activation antigen (P60), is a type II transmembrane glycoprotein with a C-type lectin-binding domain encoded by the CD69 gene located on chromosome 12 (Borrego et al, 1999). NK cells can express CD69 after activation by different stimuli such as phorbol 12- myristate 13-acetate (PMA), IL-2, IL-12, IFN-γ or anti-CD16 mAbs (Borrego et al, 1999). It is commonly used as quantitative measure of NK cell or T cell activation. Ligation of this molecule with activating anti-CD69 mAbs results in triggering of NK- cell-mediated cytolytic activity (Borrego et al, 1999). However, this activity can be abrogated by simultaneous stimulation of CD94/NKG2A inhibitory receptors.
2B4 is a glycoprotein that belongs to the signalling lymphocyte activation molecule (SLAM) family of receptors (Velikovsky et al, 2007). It is expressed on NK cells, monocytes, basophils, TCRγδ+
T cells, and a subset of CD8+ T cells (Roda- Navarro et al, 2004). Engagement of 2B4 by mAb induces its phosphorylation and recruitment of SLAM-associated protein (SAP) through cytoplasmic tyrosine-based motifs (Chen et al, 2004). The ligand of 2B4 is CD48, which is expressed on hematopoietic cells (Bryceson et al, 2006). 2B4 may function as a co-receptor in human NK-cell mediated cytotoxicity, leading to induction of cytokine (IFN-γ) and matrix metalloproteinase (MMP-2) production (Chuang et al, 2001).
43 1.2 Interleukin-6 (IL-6)
Interleukin-6 (IL-6) together with other interleukins such as IL-1 and IL-10 are pleiotropic in their effect (Kishimoto, 2006; Apte and Voronov, 2002; Commins et al, 2008). However, the IL-6 is considered the prototypic pleiotropic cytokine among them. This is reflected in the variety of names originally assigned to IL-6 based on its various functions. These include interferon beta 2 (IFN-B2) (Zilberstein et al, 1986), B cell stimulatory factor- 2 (BCSF-2) (Hirano et al, 1985; Hirano et al, 1986), B-cell differentiation factor (BCDF) (Kishimoto, 1989), plasmacytoma / hybridoma growth factor (P/HGF) (Van Damme et al, 1987; Van Snick et al,1988), 26 kDa protein (Haegeman et al, 1986), hepatocyte stimulating factor (HSF) (Baumann et al, 1987), monocyte-granulocyte inducer type 2 (MGI-2) (Shabo et al, 1988), and cytotoxic T- cell differentiation factor (CDF) (Uyttenhove et al, 1988; Ming et al, 1989). Sequencing the cDNA clones have shown that all these molecules were identical and are encoded by a single gene (Poupart et al, 1987). The name IL-6 was proposed for this molecule in December 1988 (Shegal et al, 1989; Sehgal et al, 1995). This protein has been proven to play important roles in acute phase reactions (Heinrich et al, 1990), inflammation (Hodge et al, 2005), haematopoiesis (Rodríguez et al, 2004), bone metabolism (Ishimi et al, 1990; de la Mata et al, 1995) , and cancer progression (Hodge et al, 2005; Bomberg and Wang, 2009).
1.2.1 Structure of IL-6
IL-6 is a member of a family of cytokines called the IL-6 cytokine family. This family also includes interleukin 11 (IL-11), interleukin 27 (IL-27), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), cardiotrophin-like cytokine (CLC), leukemia inhibitory factor (LIF), oncostatin M (OSM) and Kaposi's sarcoma
44 associated herpes virus interleukin-6 like protein (KSHV-IL6) (Kishimoto et al, 1995; Taga and Kishimoto, 1997). All known members of the IL-6 cytokine family have similar helical structure (Somers et al, 1997), induce hepatic expression of acute phase proteins (Somers et al, 1997) and signal through transducing receptors containing the gp 130 subunit (Boulanger et al, 2004).
Human IL-6 cDNA encodes a polypeptide of 212 amino acids with 28 amino acids as signal sequence and a 184 amino acids as mature segment (Hirano et al, 1986; Simpson et al, 1997; Li et al, 2004). The mature protein is N-glycosylated at positions 73 and 172 respectively. Due to different degrees of glycosylation and phosphorylation, IL-6 has a molecular weight ranging between 21-28 kDa (Park and Philinger, 2007). An alternative splice variant of IL-6 was identified in monocytes and lymphocytes (Kestler et al, 1995). This form is 17 kDa (148 amino acids long) and appears to lack a binding site for the IL-6 signal transducing molecule gp 130. The human IL-6 molecule contains four conserved cysteine residues involved in the formation of two disulphide bonds (Boulanger et al, 2003). A region containing nine amino acids between two of these cysteine residues are identical which led to the proposition that these cysteines were essential for the IL-6 activity. However, the biological activity of recombinant IL-6 lacking the cysteine residues suggests that these cysteines may be not necessary (Park and Philinger, 2007). Mouse and rat IL-6 also have been cloned and are approximately 65% identical to human IL-6 at cDNA level and about 40% identical at the protein level (Chiu et al, 1988; Van Snick, 1990). Unlike human IL-6, mouse and rat IL-6 lack potential N-linked glycosylation sites but may be O-glycosylated (Van Snick, 1990). Although human IL-6 is active on mouse and rat cells, mouse IL-6 is not active on human cells (Chiu et al, 1988; Northemann et al, 1989; Hammacher et al, 1994).
45 1.2.2 Gene structure
The human IL-6 genomic DNA segment has a length of approximately 5 kb and contains five exons and 4 introns (Li et al, 2004; Park and Philinger, 2007). It maps to human chromosome 7p14 and contains a c-fos serum responsive enhancer element and sequences for cAMP induction, activator protein-1 binding and glucocorticoid –responsive elements (Park and Philinger, 2007). The wide range of regulatory pathways involved in controlling IL-6 transcription confirms the central role of IL-6 in inflammatory responses. The murine gene also consists of five exons and 4 introns, but it maps to chromosome 5 (Tanabe et al, 1988). The comparison between human and mouse IL-6 genomic segments reveals highly homologous regions extending approximately 350 bp upstream of the cap site (Tanabe et al, 1988). The nucleotide sequences of IL-6 and GM-CSF genes also resemble each other in a way suggesting a possible evolutionary relationship. Four polymorphisms in the IL-6 gene have been described, with possible functional consequences (Brull et al, 2001). For example the common G C polymorphism at position -174 results in lower stimulated IL-6 promoter activity in vitro and lower circulating IL-6 concentrations in healthy individuals compared with the G allele (Fishman et al, 1998). Ferrari et al (2001) demonstrated that postmenopausal women homozygous for the -174 C allele have low serum levels of C-terminal cross-linking of type I collagen (sCTx), a marker of bone resorption.