Interferon y (IFNy)

1.2.3.1 The IFNy Gene

The IFNy gene is composed of 4 exons separated by 3 introns and is located on chromosome 12 at 12q14. The mechanisms used for IFNy transcription are well studied. Expression of the gene is correlated with hypomethylation of CG islands located in the first intron and promoter region. The promoter region spans an area of approximately 500bp (Ciccarone et a!., 1990). There also appears to be two elements in the promoter region that are essential for induction of the gene by PMA and ionomycin. Furthermore, the first intron also contains other elements that enhance transcription such as c-Rel and NF-kB sites. There are also consensus sites for STAT1, STAT4, STATS and STAT6 (Billiau, 1996). There are also several areas that bind nuclear repressors such as the silencer element a t-251 to -215, which binds yin-yang-1 (YY-1) (Ye et a!.,

1994). There are also two CREB/AP-1 sites in the promoter region that confer dexamethasone specific inhibition (Billiau, 1996). There appears to be two pathways that activate the IFNy promoter. Transcription of IFNy

can be induced either by signals from the T cell receptor or by IL-12 and IL-18 acting together (Yang etal., 1999).

1.2.3.2 The IFNy Molecule

The biologically active form of IFNy is a 34kDa homodimer although its size can vary depending upon the extent of glycosylation. The gene encodes a 166 amino acid peptide with a 23 amino acid signal peptide. The mature form is thus 143 amino acids (Billiau, 1996).

1.2.3.3 Background

The term interferon refers to the phenomenon of ‘interference’, whereby virus-infected cells are protected against further infection from other types of virus. This antiviral activity is now known to be manifested by a group of proteins known as the interferons. Type I interferons (interferons a and P) are secreted by virus infected cells, whereas type II interferon or ‘immune’ interferon is secreted by mitogen-stimulated leukocytes.

Immune interferon, now known as interferon-y, was first described over 35 years ago, when Wheelock (1965) demonstrated interferon-like anti-viral activity in the supernatants of mitogen-stimulated mononuclear cells. Later it was realised that the activity of macrophage activating factor (MAP) was also accounted for by IFNy (Nathan et a!., 1983). MAF factor was described following the discovery of the ability of mitogen-stimulated mononuclear cell cultures to enhance the activities of macrophages (Bloom & Bennet, 1966).

In vivo, IFNy is produced primarily from either T cells or NK cells. The types of T cells that produce IFNy are activated CD8+ I cells or specific subsets of CD4+ T cells (ThO or Th1) (Boehm et a!., 1997). Production of IFNy from I cells requires the activation of the T cell receptor via antigen in the context of MHC and appropriate co-stimulation. Conversely, production from NK cells does not require MHC-restricted antigen presentation and forms part of the innate immune response. The stimulus for IFNy production from NK cells comes from IL-12 and TNF produced by macrophages after having been stimulated by bacterial products (Chan at

a/., 1992).

1.2.3.4 Antigen processing/presentation

One of the earliest documented effects of IFNy was its ability to upregulate MHC class II molecules, a mechanism by which this cytokine enhances CD4+ T cell responses (Billiau, 1996). Apart from upregulating class II on APCs, it induces the de novo synthesis of class II in other cells types, therefore creating non-professional APCs. Additionally, it is also able to enhance the expression of class I molecules and p-microglobulin. This cytokine also affects antigen processing through its action of enhancing the expression of a variety of proteins needed for the process such as the transporters, TAPI and TAP2 (Epperson et a!., 1992). IFNy also modifies the repertoire of peptides that are presented by class I, as it affects changes in the composition of the subunits of the proteasome, by altering the expression of these subunits (Groettrop eta!., 1996).

1.2.3.5 Anti-viral effects

All of the interferons have anti-viral activity. The type I interferons have unique mechanisms to enable cells to resist viral infection. There are also several measures employed by both interferon types. IFNy in common with the type I interferons is able to induce several endogenous enzymes that protect against viral infection by inhibiting protein synthesis. In addition, they are able to upregulate proteins that inhibit the enzymes that are involved in driving the cell cycle.

1.2.3.6 Action on B cells

IFN-y can augment or reduce immunoglobulin secretion and may inhibit or promote the differentiation/proliferation of B cells. These effects are dependent on the state of differentiation of the B cell. Furthermore, IFN-y has an important effect of being able to induce class switching of the immunoglobulin heavy chain. IFN-y inhibits the production of IgE isotypes (mediators of hypersensitivity & allergic reactions) whilst favouring the production of lgG2a and lgG3, both of which facilitate antibody dependent cellular cytotoxicity (ADCC) (Boehm etal., 1997).

1.2.3.7 Macrophage activation

IFNy is essential in enabling the innate immune response through its action on macrophages. It increases their capacity in killing infected and neoplastic targets. Subsequent to activation by IFN-y, macrophages demonstrate an enhanced capacity for phagocytosis via complement and lgG2a receptors. The mechanisms by which macrophages kill their targets are via the use of reactive oxygen species and nitric oxide, both of which are induced by IFNy (Kaplan & Schreiber, 1999). This cytokine also controls the production of several chemokines. Induced macrophage IP- 10 is able to recruit monocytes and activated T cells and encourages T cell adhesion to endothelial cells (Taub at a!., 1993). In contrast, it inhibits the induction of IL-8 (Gusella at a!., 1993).

1.2.3.8 IFNy Polymorphism

Within the first intron of the IFNy gene a microsatellite, consisting of a variable number of CA repeats, gives rise to six alleles (Gray & Goeddel, 1982). Using PBMGs stimulated with concanavalin A, Pravica et al.

(1998) showed a significant correlation between increased in vitro IFNy production and the presence of allele 2 of this microsatellite. Subsequently this microsatellite has been associated with a SNP at the 5’ end of the CA repeat region in the first intron of the IFNy gene (+874 T/A) that lies in a putative NF-kB binding site. There was a complete correlation between the presence of the T allele and allele 2 of the microsatellite (Pravica et ai., 2000). However, a separate group have reported no association between the IFNy microsatellite and IFNy levels (Cartwright et ai., 1999).