**7.2. IL-12**

et al. [126] showed that another one of the activated STAT-responsive genes in mammary epithelial cells was OSMR. This finding was critical for completing the circle which showed that activation of OSMRβ was central to the upstream activation of the OSM-mediated path‐ way as well as to the downstream increase in the expression of the OSMRβ gene, both

The role played by activated STAT proteins in various aspects of autoimmune diseases and in oncogenesis is best exemplified by the many genes and transcription factors that have been shown to be STAT protein-responsive [51, 127]. Many of these STAT-regulated genes include additional pro-inflammatory cytokine and cytokine receptor genes besides those previously discussed. In this section we will analyze the contributions of these cytokine and

IL-18 is structurally similar to IL-1 and the IL-18 receptor is a member of the IL-1R/TLR pro‐ tein superfamily [128]. However, the function of IL-18 differs considerably from that of IL-1 and, in fact unlike IL-1, IL-18 is produced by a variety of immune as well as non-immune cells. Although IL-18 in its role as a stimulator of Th1 responses is well known by its activity as an immune defense cytokine against microbial infection, the over-production of IL-18 can result in autoimmune disease via its capacity to modify and accentuate adaptive immuno‐ logical responses such as those seen in RA [129-132]. However, paradoxically IL-18 can also stimulate Th2-related cytokine responses as well [128]. Thus, its putative role in altering the

Particularly important with regard to the role played by IL-18 in RA were results of a study by Gracie et al. [133] who first identified abundant IL-18 in RA synovial tissue. These find‐ ings are relevant when coupled with those from other studies by Tanaka et al. [134] who al‐ so found elevated IL-18 and the IL-18 receptor α/β in RA synovial tissue. They also demonstrated that IL-18 was a co-factor and regulatory cytokine in stimulating the synthesis of IFN-γ by T-cells in RA synovial tissue, the latter also requiring IL-12, thus implicating the up-regulation of IL-18 gene expression as an important component of RA disease progres‐

Activated STAT3 was identified as the JAK/STAT-related transcription factor responsible for the increased synthesis of IL-18 [127]. In that regard, TNF-α was shown to increase IL-18 gene expression in RA synoviocyte cultures suggesting the possibility that TNF-α, a known activator of p38 kinase and JNK may also activate STAT3 in synoviocyte and chondrocyte cultures. Indeed, recent results from our laboratory showed that recombinant human TNF-α activated STAT3 in normal human chondrocyte cultures and TNF-α activated STAT3, p38 kinase and JNK in cultured chondrocytes derived from human osteoarthritic knee cartilage [Malemud et al. submitted]. Thus, it was instructive to learn that treating RA patients with

events involving STAT proteins.

**7.1. IL-18**

384 Drug Discovery

sion.

**7. Other cytokines/cytokine receptors**

cytokine receptor genes to the pathology of RA.

Th1/Th2 cytokine repertoire cannot be dismissed.

IL-12 is made up of 2 disulfide-linked protein subunits, termed IL-12p35 and IL-12p40 linked in a heterodimer configuration [137, 138]. Whilst the IL-12p40 subunit has structural similarities with cytokine receptors, the IL-12p35 component is structurally similar to IL-6 and granulocyte-colony stimulating factor (G-CSF) [139]. Of note, if IL-12p35 and IL-12p40 are produced by the same cell, the bioactive heterodimer is termed, IL-12p70 [140].

IL-12 is synthesized by many cell types of the innate and adaptive immune systems, includ‐ ing, monocytes, macrophages, dendritic cells and neutrophils. IL-12 is a minor product of Bcells [140]. Although IL-12p35 is constitutively expressed at low levels by many of these cells, the expression of IL-12p40 is limited to those phagocytic cells that synthesize IL-12p70.

The connection between IL-12 and activation of the JAK/STAT pathway stems from the find‐ ing that IL-12 production was positively regulated by IFN-γ, the latter cytokine which is al‐ so induced by IL-12. Thus, IFN-γ regulates IL-12 gene expression and vice versa. By contrast, two of the anti-inflammatory cytokines, namely, IL-10 and IL-13 which also acti‐ vate JAK/STAT, suppressed IL-12 production [140] (Table 1). In addition, the type I interfer‐ on proteins, exemplified by IFN-β, which activates STAT1 [141] was shown to inhibit IL-12 gene expression in mice [142].

The main immune functions of IL-12 involve the regulation of Th1 differentiation via the ac‐ tivation of STAT4 which induces the synthesis of the T-bet transcription factor [143]. T-bet was shown to regulate IFN-γ expression and CD8+ suppressor T-cell development which had been characterized as principally IFN-γ/STAT1-dependent, and IL-12/STAT4 independ‐ ent. In fact, expression of T-bet was shown to require activated STAT4 to achieve total IL-12 dependent Th1 cell-fate determination [143]. However, Yang et al. [144] showed that the effect of IL-12/STAT4 was more complex. Thus, IL-12 -induced activated STAT4 bound to a distant but highly conserved STAT-responsive T-bet enhancer region where it induced IFNγ-activated STAT1 independent T-bet gene expression in CD8+ cells. Importantly, IL-4-in‐ duced STAT6 activation regulates the development and effector functions, not of Th1 cells, but rather of Th2 cells in peripheral tissues such as skin, lung and gut [145]. However, Th2 cell produced in lymph nodes did not require IL-4-mediated activation of STAT6 [145].

In summary, cell-fate determination induced by the IL-12-mediated activation of STAT4, IL-4-mediated activation of STAT6, transforming growth factor-β (TGF-β), IL-6 plus TGF-β and IL-27 activation of STAT3 profoundly influence the balance of Th1 and Th2 cells, Th17 cells and Treg cell production, respectively [80, 146-149]. This conclusion must, however, be tempered by results of recent studies which also showed that formal interplays occurred be‐ tween IL-4-induced STAT6 phosphorylation, the GATA-binding protein-3 (GATA3) zinc-fin‐ ger transcription factor [150] and the Treg cell transcription factor, FoxP3 as well. Importantly, GATA3 was revealed as the key transcription factor in this complex interplay because GATA3 could 1) directly inhibit Th1 differentiation through its capacity to block upregulation of the IL-12β2 receptor; 2) inhibit the activity of STAT4; and 3) neutralize the ac‐ tivity of runt-related transcription factor 3 (runx3), via its capacity to induce protein-protein interactions [150]. Thus, by modulating the activities of IL-4/STAT6, GATA3/STAT4 and runx3 one could potentially alter the activity of pro-inflammatory and anti-inflammatory cy‐ tokines as well as overcome immune tolerance.

Implying a role for IL-21 in the development and progression of RA would also depend on finding an elevated level of IL-21 in human RA tissues and by demonstrating an involve‐ ment of IL-21 in the pathogenesis of CIA or inflammatory arthritis in other animal models. Thus the results of a study by Young et al. [158] were noteworthy in this regard for several reasons. First and foremost, treating DBA mice with CIA with an antibody to IL-21R (i.e. IL-21R.Fc) reduced the severity of arthritis. The reduction in hind paw swelling was accom‐ panied by lower levels of IL-6 in the hind paw but also in the sera of mice treated with IL-21R.Fc suggesting that one of the downstream events regulated by IL-21 was IL-6 gene expression. Of note, the level of INF-γ was increased in the hind paws of mice with CIA. Furthermore, the cultured cells from the lymph nodes of mice with CIA treated with IL-21R.Fc showed an increased level of IFN-γ *ex vivo*. These findings (i.e. reduced IFN-γ; in‐ creased IL-6) were mirrored *ex vivo* using Type II collagen-specific spleen cells from CIA mice treated with IL-21R.Fc. Most importantly from the perspective of potentially using an anti-IL-21R antibody as a therapeutic agent for RA was the finding that treating Lewis rats with adjuvant –induced arthritis therapeutically with IL-21R.Fc "reversed" the swelling in inflamed joints and tissues from these joints whilst the tissues showed improvement using a well-validated histological scoring system. More recently, Yuan et al. [159] showed that IL-21R mRNA was found in human RA synovial tissue samples. In addition, this group also confirmed the results of the Young et al. study [158] since they showed that an anti-IL-21R antibody ameliorated the severity of arthritis in CIA which was accompanied by reduced cytokine levels in cells derived from the anti-IL-21R antibody-treated mice. Interestingly, IL-21R-deficient K/BxN mice [160] failed to develop arthritis; a result which suggested that

Suppression of Pro-Inflammatory Cytokines via Targeting of STAT-Responsive Genes

http://dx.doi.org/10.5772/52506

387

IL-21R played a critical role in the pathogenesis of K/BxN serum-induced arthritis.

pro-inflammatory cytokine levels in RA.

**8. The extended IL-10 cytokine superfamily**

which now comprise the IFN-λ cytokine subfamily [164-166].

There now are several lines of evidence that showed that the IL-21/IL-21R pathway plays a functional role in regulating inflammatory responses in autoimmune arthritis. In that re‐ gard, anti-IL-21 blockade should also be considered for future drug development for RA. However, what would also be crucial to improving our understanding of the role of IL-21 in RA would be to discover which pro-inflammatory cytokine levels are altered in response to the JAK/STAT activation by IL-21/IL21R. This could provide a novel paradigm for reducing

IL-19, IL-20, IL-22, IL-24 (melanoma differentiation-associated gene 7; mda-7), and IL-26 (AK155) are all structurally similar to IL-10 and these interleukins constitute members of the extended IL-10 cytokine superfamily [161-163]. Three additional members of the IL-10 cyto‐ kine superfamily have recently been added to this list, namely, IL-28A, IL-28B and IL-29

IL-19 and IL-20 are α-helical proteins. They have similar cysteine sites; their amino acid se‐ quences are approximately 30% identical. In the human genome, the genes encoding these IL-10 superfamily members are located in two clusters; one cluster comprises the genes for
