**3. A special look at the pathogenetic functions of keratinocytes**

Besides erythema, impressive hyperkeratosis manifesting as large, silvery scales, is clinically the most visible pathology and represents a hallmark of psoriasis. This typical and characteristic epidermal involvement has in the past led to discussions on whether hyperproliferation and altered differentiation of epidermal keratinocytes occur indeed only in response to skin inflammation or whether keratinocytes themselves have their share in initiation and/or propagation of psoriasis. Whereas it is widely accepted today that keratinocytes have the potential to actively participate and modulate immune reactions in the skin their role as initiators or amplifiers of the inflammatory reaction in psoriasis is still not so clear (Tschachler, 2007). Some evidence indicates that the exposure of altered autoantigens by keratinocytes could be directly responsible for the activation and expansion of certain T-cell subpopulations in psoriatic skin (Bos et al., 2005). A keratinocyte-derived candidate auto-antigen is keratin 17. Patients with active psoriasis have an increased frequency of circulating Th1 cells reacting to peptides from keratin 17 that shares ALEEAN aminoacidic sequence with the streptococcus M-protein. Using a new approach termed SErological identification of Recombinant EXpressed antigens (SEREX), new auto-antigens were found in the serum of patients with psoriasis (Jones et al., 2004). Keratin 13, heterogeneous ribonucleprotein-A1, and a previously uncharacterized protein, FLJ00294, were identified by SEREX as representative antigens in psoriatic patients, although autoreactivity for these proteins was also detected in control subjects without psoriasis. Keratinocytes could be indirectly responsible for the activation of pathogenetic T cells through the exposure to viral or bacterial products. Under the influence of IL-17 and IL-22, keratinocytes, are able to produce AMPs like human beta defensin 2, and S100 proteins (Nograles et al., 2008). The expression of another antimicrobial peptide, LL-37 cathelicidin, can also be enhanced by IL-17 in the presence of vitamin D3 (Peric et al., 2008). These proteins may function as key inflammation inducers in psoriasis, and at the same time decrease skin infections under conditions of a dysfunctional epidermal barrier. Infections or injury to the skin can promote lesion formation in susceptible individuals and these triggers have been shown to stimulate keratinocyte production of the antimicrobial LL-37 cathelicidin that, when complexed with self-DNA, binds to TLR9 on plasmocytoid DCs. These cells produce massive amounts of IFN-α and are implicated in the initiation of psoriasis lesions (Lande et al., 2007). Accordingly, patients treated with a topical plasmocytoid DCs agonist, imiquimod, up-regulate IFN-α and experience exacerbations in psoriasis. In addition to stimulating plasmocytoid DCs, LL-37 has been shown to complex with self-RNA to trigger the activation of myeloid dendritic cells (myeloid DCs) through TLR8. This leads to production of TNF-α and IL-6, and promotes their differentiation into mature dendritic cells (Ganguly et al., 2009). Because myeloid DCs in psoriasis have been shown to produce IL-23 it is plausible that self-RNA complexes might potentially initiate the inflammatory cascade leading to expansion and activation of Th17 cells (Nograles et al., 2010).

Recent investigations identified high levels of osteopontin (OPN) in psoriatic plaques (Buommino et al., 2009). Osteopontin is produced by both keratinocytes and activated T cells. It is a phosphorylated acidic glycoprotein of pleiotropic properties and has been recently recognized as a potential inflammatory cytokine. A model for the role of OPN in Th1 Th17 psoriatic disease was so suggested. After activation of myeloid DCs that express OPN, they migrate to skin draining lymph nodes and polarize naive T cells towards a Th1 and Th17 phenotype. In addition OPN secreted by keratinocytes attracts additional inflammatory cells. Moreover OPN inhibits keratinocyte apoptosis thereby supporting enhanced epidermal proliferation, and, through a pro-angiogenic effect on microvascular endothelial cells, OPN also promotes vessel formation subsequently supporting the influx of inflammatory cells (Buback et al., 2009).

#### **3.1 Keratinocytes and cytokines**

12 Psoriasis

operating between keratinocytes and fibroblasts that culminates with keratinocyte proliferation is triggered by IL-1: IL-1α and IL-1β neutralization and IL-1 receptor antagonist significantly reduced keratinocyte growth through the abrogation of keratinocyte growth factor (KGF) production by fibroblasts. However, IL-1 is unlikely to be the only regulator of KGF production by fibroblasts, and indeed other keratinocyte-derived factors, such as parathyroid hormone–related protein (PTHrP), induce KGF expression. In addition fibroblast growth factor (FGF) family members and granulocyte-macrophage colony stimulating factor (GM-CSF) also play a particularly important role in the fibroblast-driven regulation of keratinocyte proliferation (Albanesi et al., 2007). Moreover, the activated phenotype of lesional endothelial cells are believed to play a central role in the pathogenesis of psoriasis and are determined by the expression of a variety of membrane and soluble factors mainly responsible for T-cell recruitment in the skin like adhesion molecule 1 (ICAM-1). A key point is the endothelium expression of certain chemokines involved in the arrest of circulating T lymphocytes at inflammatory sites: upon exposure to inflammatory signals, mainly represented by TNF-α and IL-1, endothelial cells express a broad array of chemokines, including CCL20/MIP-3α, CXCL12/SDF-1, CCL21/SLC, CCL17/TARC, CCL2/MCP-1, CXCL10/IL-8, CCL5/RANTES, CXCL1/Gro-α, and CCL4/MIP-1β

In summary, feedback loops involving keratinocytes, fibroblast and endothelial cells contribute to tissue reorganization with endothelial cell activation and proliferation and deposition of extracellular matrix. The hypothesis of cytokine/chemokine network in

Besides erythema, impressive hyperkeratosis manifesting as large, silvery scales, is clinically the most visible pathology and represents a hallmark of psoriasis. This typical and characteristic epidermal involvement has in the past led to discussions on whether hyperproliferation and altered differentiation of epidermal keratinocytes occur indeed only in response to skin inflammation or whether keratinocytes themselves have their share in initiation and/or propagation of psoriasis. Whereas it is widely accepted today that keratinocytes have the potential to actively participate and modulate immune reactions in the skin their role as initiators or amplifiers of the inflammatory reaction in psoriasis is still not so clear (Tschachler, 2007). Some evidence indicates that the exposure of altered autoantigens by keratinocytes could be directly responsible for the activation and expansion of certain T-cell subpopulations in psoriatic skin (Bos et al., 2005). A keratinocyte-derived candidate auto-antigen is keratin 17. Patients with active psoriasis have an increased frequency of circulating Th1 cells reacting to peptides from keratin 17 that shares ALEEAN aminoacidic sequence with the streptococcus M-protein. Using a new approach termed SErological identification of Recombinant EXpressed antigens (SEREX), new auto-antigens were found in the serum of patients with psoriasis (Jones et al., 2004). Keratin 13, heterogeneous ribonucleprotein-A1, and a previously uncharacterized protein, FLJ00294, were identified by SEREX as representative antigens in psoriatic patients, although autoreactivity for these proteins was also detected in control subjects without psoriasis. Keratinocytes could be indirectly responsible for the activation of pathogenetic T cells through the exposure to viral or bacterial products. Under the influence of IL-17 and IL-22, keratinocytes, are able to produce AMPs like human beta defensin 2, and S100 proteins

psoriasis proposed a central role of pro-inflammatory cytokines, including TNF-α.

**3. A special look at the pathogenetic functions of keratinocytes** 

(Girolomoni et al., 2004).

Cutaneous and systemic over-expression of various pro-inflammatory cytokines has been demonstrated in psoriasis. Psoriatic keratinocytes are able to produce and release IL-1α, IL-1β, IL-6, IL-15, IL-18 and IL-20, all of them involved in the development of different alterations which compose the complex and intricate net of psoriasis pathogenesis (Tab. 1).The cellular composition of the inflammatory infiltrate within the psoriatic plaques as well as hyperproliferation of keratinocytes and so the whole pathogenetic process of psoriasis appears to be mediated by these cytokines (Wojas-Pelc et al., 2006).

#### **3.1.1 Keratinocytes and IL-1α & IL-1β**

IL-1 is a pro-inflammatory cytokine stimulating, among others, IL-2 and IFN-γ production through activated T cells. IL-1 activates neutrophils, monocytes, eosinophils and basophils, triggers production of TNF-α, IL-6, IL-8 by macrophages, and in autocrine fashion, IL-1 synthesis. IL-1 promotes proliferation of bone marrow cells, B lymphocytes, neutrophils, macrophages and platelets (Dinariello, 2002). In psoriasis, keratinocytes are the main source of IL-1α and IL-1β in the skin stored in the form of precursor particles (Zepter et al., 1997). Monocytes/macrophages, activated endothelial cells, fibroblasts and

Pathogenesis of Psoriasis: The Role of Pro-Inflammatory Cytokines Produced by Keratinocytes 15

IL-6 is involved in the growth and differentiation of dermal and epidermal cells (Hirano, 1998), growth and differentiation of cytotoxic cells, activation of natural killer cells (NK) and maturation of hematopoietic stem cells (Pietrzak et al., 1999). Furthermore it acts as a chemotactic factor for T cells, and thus can directly stimulate T-cell migration to the epidermis. Increased levels of mRNA of IL-6 and its receptor were observed in psoriatic lesions, suction blister fluid and in keratinocytes (Krasowska et al., 1998). Previous studies have also shown a high level of IL-6 in plasma/serum of patients with psoriasis (Galadari & Sheriff, 2005; Grossman et al., 1989). Higher IL-6 levels were observed in psoriatic lesions compared to non-lesional and normal healthy skin (Chang et al., 1992; Grossman et al., 1989). Classical anti-psoriatic therapies such as phototherapy (PUVA, UVB), systemic corticosteroids and methothrexate lead to normalization of IL-6 levels (Mizutani et al., 1997). Both non-lesional and lesional psoriatic keratinocytes produce IL-6 (Grossman et al., 1989; Krasowska et al., 1998; Zalewska et al., 2006). IL-1 and TNF-α activate keratinocytes to produce IL-6. Koebner phenomenon is likely to result from the increased activity of IL-6 and its receptor in psoriasis (Grossman et al., 1989). Many studies show that IL-17F is able to induce IL-6 production both in normal human epidermal keratinocytes and in mouse skin (Fujishima et al., 2010). Moreover CD4+ T cells in skin from psoriasis patients express IL-17F and recent studies have demonstrated increased expression of IL-6 in IL-17F-overexpressing mice, thus further supporting a role of IL-17F in the induction of IL-6 (Hurst et al., 2002; Yang et al., 2008). IL-6 could directly contribute to the epidermal hyperplasia seen in psoriatic epithelium as well as affecting the function of dermal inflammatory cells. Moreover, it has been demonstrated that IL-6 induces Th17 cell differentiation in humans (Ishigame et al., 2009). Taken together, all these data suggest that IL-17F-induced IL-6 produced by keratinocytes promotes the development of Th17 cells as an autocrine regulator. Thus, the IL-17F/IL-6 axis may enhance inflammation of the lesional skin in

IL-15 is a pro-inflammatory cytokine involved in chronic inflammatory processes. It is a key factor controlling the activation, proliferation and survival of NK cells (Fehniger & Caligiuri, 2001; Liu et al., 2000). IL-15 is also a strong chemotactic factor for leukocytes. This cytokine triggers angiogenesis and exerts strong anti-apoptotic effects, especially on lymphocytes, hepatocytes and keratinocytes (Berard et al., 2003; Rückert et al., 2000). Furthermore, it stimulates the expression of IL-17 by T cells (Elder, 2007). Elevated levels of IL-15 were noted in the lesional psoriatic skin (Elder, 2007; Rückert et al., 2000; Yano et al., 2003). Monocytes and macrophages represent the main source of IL-15 (Fehniger & Caligiuri, 2001; Musso et al., 1999) in lesional psoriatic skin, as well as keratinocytes (McInnes & Gracie, 2004; Yano et al., 2003). Lesional keratinocytes are strong producers of IL-15, which not only appears critical in the promotion of T cell and monocyte activation and, hence, in the maintenance of the local pro-inflammatory milieu, but also in the keratinocyte self-protection from apoptosis (Rückert et al., 2000); the pathogenic effect of this cytokine in psoriasis probably results from the stimulation of proliferation and activation of T cells and pro-inflammatory cytokines release (including TNF-α). Recent genetic studies (Elder, 2007) further supported the role of IL-15 as an important factor in psoriasis pathogenesis: IL-15 acts as a growth factor for CD8+ T cells, which infiltrate the epidermis during the development of psoriatic lesions, triggers

**3.1.2 Keratinocytes and IL-6** 

psoriasis (Fujishima et al., 2010).

**3.1.3 Keratinocytes and IL-15** 

Langerhans cells (LCs) are additional IL-1 sources (Yoshinaga et al., 1995). Normal keratinocytes do not contain a biologically active form of interleukin 1β-converting enzyme (ICE), and almost all IL-1 activity in the healthy epidermis results from the activity of IL-1α. In transgenic mouse models, IL-1α production in the basal layer of the epidermis leads to development of inflammatory lesions characterized by erythema and histology resembling psoriasis (Groves et al., 1995). Although IL-1 expression in the psoriatic epidermis appears altered, data on this finding are often conflicting. Some studies showed that IL-1α levels in psoriatic lesions were decreased or below detection limits in comparison to non-lesional and healthy skin (Okubo & Koga, 1998), whereas others demonstrated increased levels of IL-1β (Debets et al., 1997). Serum levels of IL-1α and IL-1β were low both in patients and in healthy controls. Increased levels of IL-1α and IL-1β were noted in supernatants of monocyte cultures obtained from patients with psoriasis (Okubo & Koga, 1998). Peripheral blood mononuclear cells (PBMCs) of inactive psoriasis patients produced lower levels of IL-1α and IL-1β than the cells obtained from patients with active psoriasis, although still higher than those of healthy controls. The production of IL-1β by PBMCs from psoriatic patients positively correlated with disease severity (Mizutani et al., 1997). Higher levels of IL-1β in blister fluid than in serum support the hypothesis that this cytokine is locally produced in psoriatic lesions. Despite fairly strong arguments for the key role of IL-1 in the activation of psoriasis, there is scarcity of data on the use of IL-1 antagonists in psoriasis treatment.



#### **3.1.2 Keratinocytes and IL-6**

14 Psoriasis

Langerhans cells (LCs) are additional IL-1 sources (Yoshinaga et al., 1995). Normal keratinocytes do not contain a biologically active form of interleukin 1β-converting enzyme (ICE), and almost all IL-1 activity in the healthy epidermis results from the activity of IL-1α. In transgenic mouse models, IL-1α production in the basal layer of the epidermis leads to development of inflammatory lesions characterized by erythema and histology resembling psoriasis (Groves et al., 1995). Although IL-1 expression in the psoriatic epidermis appears altered, data on this finding are often conflicting. Some studies showed that IL-1α levels in psoriatic lesions were decreased or below detection limits in comparison to non-lesional and healthy skin (Okubo & Koga, 1998), whereas others demonstrated increased levels of IL-1β (Debets et al., 1997). Serum levels of IL-1α and IL-1β were low both in patients and in healthy controls. Increased levels of IL-1α and IL-1β were noted in supernatants of monocyte cultures obtained from patients with psoriasis (Okubo & Koga, 1998). Peripheral blood mononuclear cells (PBMCs) of inactive psoriasis patients produced lower levels of IL-1α and IL-1β than the cells obtained from patients with active psoriasis, although still higher than those of healthy controls. The production of IL-1β by PBMCs from psoriatic patients positively correlated with disease severity (Mizutani et al., 1997). Higher levels of IL-1β in blister fluid than in serum support the hypothesis that this cytokine is locally produced in psoriatic lesions. Despite fairly strong arguments for the key role of IL-1 in the activation of psoriasis, there is

scarcity of data on the use of IL-1 antagonists in psoriasis treatment.

IL-1 Stimulation of IL-2 and IFN-γ production

IL-6 Regulation of growth and differentiation of

IL-15 Anti-apoptotic effects on lymphocytes and

IL-18 Induction of several chemokines in

IL-20 Inhibition of normal terminal

Table 1. Roles of cytokines released by psoriatic keratinocytes.

through activated T cells and of TNF-α, IL-6, IL-8 by macrophages, and in autocrine

epidermal cells and stimulation of Th17

cytokines implicated in the pathogenesis of psoriasis, including IFN-γ and TNF-α.

fibroblasts and neutrophils, increased Tcell adhesion to extracellular matrix ligands, induction of angiogenesis, induction of chemotaxis in plasmacytoid

differentiation of keratinocytes, induction

keratinocytes; stimulation of IL-17 expression, promotion of T cell and monocyte activation, production of

fashion, of IL-1 synthesis.

cells differentiation.

dendritic cells.

of anti-bacterial proteins.

**Cytokines Role in psoriasis** 

IL-6 is involved in the growth and differentiation of dermal and epidermal cells (Hirano, 1998), growth and differentiation of cytotoxic cells, activation of natural killer cells (NK) and maturation of hematopoietic stem cells (Pietrzak et al., 1999). Furthermore it acts as a chemotactic factor for T cells, and thus can directly stimulate T-cell migration to the epidermis. Increased levels of mRNA of IL-6 and its receptor were observed in psoriatic lesions, suction blister fluid and in keratinocytes (Krasowska et al., 1998). Previous studies have also shown a high level of IL-6 in plasma/serum of patients with psoriasis (Galadari & Sheriff, 2005; Grossman et al., 1989). Higher IL-6 levels were observed in psoriatic lesions compared to non-lesional and normal healthy skin (Chang et al., 1992; Grossman et al., 1989). Classical anti-psoriatic therapies such as phototherapy (PUVA, UVB), systemic corticosteroids and methothrexate lead to normalization of IL-6 levels (Mizutani et al., 1997). Both non-lesional and lesional psoriatic keratinocytes produce IL-6 (Grossman et al., 1989; Krasowska et al., 1998; Zalewska et al., 2006). IL-1 and TNF-α activate keratinocytes to produce IL-6. Koebner phenomenon is likely to result from the increased activity of IL-6 and its receptor in psoriasis (Grossman et al., 1989). Many studies show that IL-17F is able to induce IL-6 production both in normal human epidermal keratinocytes and in mouse skin (Fujishima et al., 2010). Moreover CD4+ T cells in skin from psoriasis patients express IL-17F and recent studies have demonstrated increased expression of IL-6 in IL-17F-overexpressing mice, thus further supporting a role of IL-17F in the induction of IL-6 (Hurst et al., 2002; Yang et al., 2008). IL-6 could directly contribute to the epidermal hyperplasia seen in psoriatic epithelium as well as affecting the function of dermal inflammatory cells. Moreover, it has been demonstrated that IL-6 induces Th17 cell differentiation in humans (Ishigame et al., 2009). Taken together, all these data suggest that IL-17F-induced IL-6 produced by keratinocytes promotes the development of Th17 cells as an autocrine regulator. Thus, the IL-17F/IL-6 axis may enhance inflammation of the lesional skin in psoriasis (Fujishima et al., 2010).

#### **3.1.3 Keratinocytes and IL-15**

IL-15 is a pro-inflammatory cytokine involved in chronic inflammatory processes. It is a key factor controlling the activation, proliferation and survival of NK cells (Fehniger & Caligiuri, 2001; Liu et al., 2000). IL-15 is also a strong chemotactic factor for leukocytes. This cytokine triggers angiogenesis and exerts strong anti-apoptotic effects, especially on lymphocytes, hepatocytes and keratinocytes (Berard et al., 2003; Rückert et al., 2000). Furthermore, it stimulates the expression of IL-17 by T cells (Elder, 2007). Elevated levels of IL-15 were noted in the lesional psoriatic skin (Elder, 2007; Rückert et al., 2000; Yano et al., 2003). Monocytes and macrophages represent the main source of IL-15 (Fehniger & Caligiuri, 2001; Musso et al., 1999) in lesional psoriatic skin, as well as keratinocytes (McInnes & Gracie, 2004; Yano et al., 2003). Lesional keratinocytes are strong producers of IL-15, which not only appears critical in the promotion of T cell and monocyte activation and, hence, in the maintenance of the local pro-inflammatory milieu, but also in the keratinocyte self-protection from apoptosis (Rückert et al., 2000); the pathogenic effect of this cytokine in psoriasis probably results from the stimulation of proliferation and activation of T cells and pro-inflammatory cytokines release (including TNF-α). Recent genetic studies (Elder, 2007) further supported the role of IL-15 as an important factor in psoriasis pathogenesis: IL-15 acts as a growth factor for CD8+ T cells, which infiltrate the epidermis during the development of psoriatic lesions, triggers

Pathogenesis of Psoriasis: The Role of Pro-Inflammatory Cytokines Produced by Keratinocytes 17

stratum corneum from living keratinocytes of the upper (granular) epidermis layer (Candi et al., 2005). In psoriatic lesions, which contain high levels of IL-20, this process is altered. Furthermore, IL-20 simultaneously enhance the K16 expression, a keratin known to be upregulated in psoriatic lesions and associated with keratinocyte regeneration (Wolk et al., 2006, 2009b). Apart from the inhibition of normal terminal differentiation of keratinocytes, IL-20 in addition to other mediators (Kanda & Watanabe, 2008), induce a state of enhanced antimicrobial defence of the epidermis by inducing a range of antimicrobial proteins (Sa et al., 2007; Wolk et al., 2004, 2006). In psoriatic lesions IL-20 expression was found preferentially in basal and supra-basal keratinocytes above the dermal papillae (Romer et al., 2003; Wolk et al., 2009a). Most interestingly, IL-17 and TNF- amplified the IL-22 induced production of IL-20 in keratinocytes. In summary, the T/NK cell cytokine IL-22 induces the keratinocyte secretion of IL-20 as a second mediator that has very similar effects to its own. IL-20, therefore, may, to some extent, further amplify and/or prolong the IL-22 action on the keratinocyte

differentiation that leads to the characteristic epidermal changes observed in psoriasis.

TNF-α is a key pro-inflammatory cytokine with an important pathogenetic role in psoriasis and psoriatic arthritis. The evidence includes further observations that a variety of anti-TNF-α approaches such as monoclonal antibodies and fusion proteins of soluble TNF-α receptors are effective therapies both in psoriasis and psoriatic arthritis. As for TNF-α itself, production of this cytokine is mainly attributed to immune cells (Lowes et al., 2007); however, it is noteworthy that keratinocytes are also able to elaborate TNF-α (Gottlieb at al., 2005). In psoriasis, the inflammatory response to TNF-α could be self-sustaining: activated dendritic cells are the major source of TNF-α in psoriasis lesions (Boyman et al., 2004) and at the same time TNF-α mRNA is induced in keratinocytes after TNF-α exposure (Gottlieb et al., 2005). Low level of TNF-α is present in the upper layer of the healthy epidermis, but its synthesis and release from keratinocytes are greatly augmented by injury, infection and UV irradiation. Of the two distinct cell-surface receptors for TNF-α, TNFR1 and TNFR2, keratinocytes mainly express TNFR1 (Kondo & Sauder, 1997). The binding of TNF-α to TNFR1 triggers a series of intracellular events resulting in the activation of transcription factors, including NF-KB, AP-1, CCAAT enhancer-binding protein-β, and others, which are responsible for the induction of genes important for diverse biological processes, including cell growth and death and immune, inflammatory, and stress responses (Banno et al., 2004). TNF-α activates the immune responses through inducing the production of additional signals, such as IL-1 and IL-8, transforming growth factor type-β (TGF-β) and ICAM-1. Psoriatic keratinocytes are also an important source of IL-7. Increased IL-7 levels were observed in both the psoriatic skin and serum of psoriatic patients (Bonifati et al., 1997; Pietrzak et al., 2008). However, no correlation between IL-7 levels and psoriasis area severity index (PASI) score was observed. In addition, IL-7 levels did not decrease after effective anti-psoriatic treatment, which suggests that this cytokine could not be regarded as a marker of the disease activity. IL-7 is a pleiotropic cytokine playing an essential role in the development and differentiation of T cells. IL-7 regulates survival, proliferation and cytotoxicity of maturation of T cells at the periphery. Furthermore, IL-7 together with IL-2 and IL-12, can induce the synthesis of IFN-γ, while, in turn, IFN-γ induces IL-7 secretion by keratinocytes (Ariizumi et al., 1995). In psoriasis IL-7 seems to play a key role in driving

**3.1.6 Keratinocytes and other cytokines** 

inflammatory cell recruitment, angiogenesis, and production of other cytokines implicated in the pathogenesis of psoriasis, including IFN-γ and TNF-α.

#### **3.1.4 Keratinocytes and IL-18**

IL-18 exerts its activity on the human defense system in inflammatory, infectious and autoimmune diseases (Dinarello, 2006). IL-18 over-production stimulates the recruitment of dendritic cells to the site of inflammation (Gutzmer et al., 2003). IL-18, especially together with IL-12, triggers the production of IFN-γ in many immunocompetent cells, including NK cells, T helper and cytotoxic cells. Subsequently, IFN-γ decreases Th2 response and enhances Th1 response by stimulating cytotoxic T cells (Ericson et al., 2004). Thus, IL-18 possesses the capacity to stimulate innate immunity as well as Th1-mediated responses (Nakanishi et al., 2001). IL-18 overproduction is characteristic for many diseases including psoriasis (Nakanishi et al., 2001). The role of IL-18 in psoriasis has not been fully elucidated. It is speculated that IL-18 produced by human keratinocytes enhances IFN-γ production in inflammation and thus IL-18 seems to be a promising target in Th1-type inflammatory diseases, like psoriasis (McKenzie et al., 2002; Ohta et al., 2001). Its expression in psoriasis is significantly enhanced in supra-basal keratinocytes (Flisiak et al., 2006; McKenzie et al., 2002; Ohta et al., 2001). Reverse transcription polymerase chain reaction (RT-PCR) revealed IL-18 mRNA levels to be two to eight times higher in psoriatic skin biopsies than in the nonlesional psoriatic skin and healthy controls. Overexpression of IL-18 was observed in keratinocytes of the whole epidermis in psoriatic lesions and in the basal layer of non-lesional epidermis compared to only slight IL-18 expression in the epidermis of healthy controls (McKenzie et al., 2002). McKenzie et al. reported six to eight-fold higher levels of the IL-18 receptor mRNA in the epidermis of psoriatic lesions compared to non-lesional and healthy control skin. Moreover total IL-18 protein levels were found to be 3.5 times higher in the active and progressive psoriatic epidermis compared to the normal and stable, plaque-type psoriatic epidermis. To date, there are only a few studies on IL-18 in the blood of psoriatic patients (Flisiak et al., 2006, Gangemi et al., 2003), which revealed increased plasma IL-18 levels in psoriatic patients in comparison to controls. IL-18 might act in the early phases of psoriasis via IFN-γ independent routes, such as: a) induction of several chemokines in fibroblasts and neutrophils (Leung et al., 2001; Morel et al., 2001); b) increased T-cell adhesion to extracellular matrix ligands (Ariel et al., 2002); c) induction of angiogenesis (Park et al., 2001); d) induction of chemotaxis in plasmacytoid dendritic cells (Kaser et al., 2004). Thus, IL-18 could be involved in the regulation of early inflammatory events by promoting the recruitment and adhesion of the immune system cells to the inflamed sites. However, whether IFN-γ-dependent or independent mechanisms are responsible for the IL-18 activity in early stages of psoriatic plaque development remains to be elucidated.

#### **3.1.5 Keratinocytes and IL-20**

IL-20 was demonstrated to promote hyperproliferation of keratinocytes by activating IL-20 receptor to modulate skin inflammation. It was also reported that IL-20 induced IL-6 and TNFα in monocytes, stimulated the expression of keratinocytes growth factor (KGF), IL-6, TNF-α and reactive oxygen species (ROS) in CD8+ T cells (Wei et al., 2006). In psoriasis, the two most important effects of IL-20 are the inhibition of normal terminal differentiation of keratinocytes and the induction of anti-bacterial proteins (Wolk et al., 2009a). Keratinocyte terminal differentiation is the apoptosis-like process that generates corneocytes for the desquaming stratum corneum from living keratinocytes of the upper (granular) epidermis layer (Candi et al., 2005). In psoriatic lesions, which contain high levels of IL-20, this process is altered. Furthermore, IL-20 simultaneously enhance the K16 expression, a keratin known to be upregulated in psoriatic lesions and associated with keratinocyte regeneration (Wolk et al., 2006, 2009b). Apart from the inhibition of normal terminal differentiation of keratinocytes, IL-20 in addition to other mediators (Kanda & Watanabe, 2008), induce a state of enhanced antimicrobial defence of the epidermis by inducing a range of antimicrobial proteins (Sa et al., 2007; Wolk et al., 2004, 2006). In psoriatic lesions IL-20 expression was found preferentially in basal and supra-basal keratinocytes above the dermal papillae (Romer et al., 2003; Wolk et al., 2009a). Most interestingly, IL-17 and TNF- amplified the IL-22 induced production of IL-20 in keratinocytes. In summary, the T/NK cell cytokine IL-22 induces the keratinocyte secretion of IL-20 as a second mediator that has very similar effects to its own. IL-20, therefore, may, to some extent, further amplify and/or prolong the IL-22 action on the keratinocyte differentiation that leads to the characteristic epidermal changes observed in psoriasis.

#### **3.1.6 Keratinocytes and other cytokines**

16 Psoriasis

inflammatory cell recruitment, angiogenesis, and production of other cytokines implicated in

IL-18 exerts its activity on the human defense system in inflammatory, infectious and autoimmune diseases (Dinarello, 2006). IL-18 over-production stimulates the recruitment of dendritic cells to the site of inflammation (Gutzmer et al., 2003). IL-18, especially together with IL-12, triggers the production of IFN-γ in many immunocompetent cells, including NK cells, T helper and cytotoxic cells. Subsequently, IFN-γ decreases Th2 response and enhances Th1 response by stimulating cytotoxic T cells (Ericson et al., 2004). Thus, IL-18 possesses the capacity to stimulate innate immunity as well as Th1-mediated responses (Nakanishi et al., 2001). IL-18 overproduction is characteristic for many diseases including psoriasis (Nakanishi et al., 2001). The role of IL-18 in psoriasis has not been fully elucidated. It is speculated that IL-18 produced by human keratinocytes enhances IFN-γ production in inflammation and thus IL-18 seems to be a promising target in Th1-type inflammatory diseases, like psoriasis (McKenzie et al., 2002; Ohta et al., 2001). Its expression in psoriasis is significantly enhanced in supra-basal keratinocytes (Flisiak et al., 2006; McKenzie et al., 2002; Ohta et al., 2001). Reverse transcription polymerase chain reaction (RT-PCR) revealed IL-18 mRNA levels to be two to eight times higher in psoriatic skin biopsies than in the nonlesional psoriatic skin and healthy controls. Overexpression of IL-18 was observed in keratinocytes of the whole epidermis in psoriatic lesions and in the basal layer of non-lesional epidermis compared to only slight IL-18 expression in the epidermis of healthy controls (McKenzie et al., 2002). McKenzie et al. reported six to eight-fold higher levels of the IL-18 receptor mRNA in the epidermis of psoriatic lesions compared to non-lesional and healthy control skin. Moreover total IL-18 protein levels were found to be 3.5 times higher in the active and progressive psoriatic epidermis compared to the normal and stable, plaque-type psoriatic epidermis. To date, there are only a few studies on IL-18 in the blood of psoriatic patients (Flisiak et al., 2006, Gangemi et al., 2003), which revealed increased plasma IL-18 levels in psoriatic patients in comparison to controls. IL-18 might act in the early phases of psoriasis via IFN-γ independent routes, such as: a) induction of several chemokines in fibroblasts and neutrophils (Leung et al., 2001; Morel et al., 2001); b) increased T-cell adhesion to extracellular matrix ligands (Ariel et al., 2002); c) induction of angiogenesis (Park et al., 2001); d) induction of chemotaxis in plasmacytoid dendritic cells (Kaser et al., 2004). Thus, IL-18 could be involved in the regulation of early inflammatory events by promoting the recruitment and adhesion of the immune system cells to the inflamed sites. However, whether IFN-γ-dependent or independent mechanisms are responsible for the IL-18 activity in early stages of psoriatic

IL-20 was demonstrated to promote hyperproliferation of keratinocytes by activating IL-20 receptor to modulate skin inflammation. It was also reported that IL-20 induced IL-6 and TNFα in monocytes, stimulated the expression of keratinocytes growth factor (KGF), IL-6, TNF-α and reactive oxygen species (ROS) in CD8+ T cells (Wei et al., 2006). In psoriasis, the two most important effects of IL-20 are the inhibition of normal terminal differentiation of keratinocytes and the induction of anti-bacterial proteins (Wolk et al., 2009a). Keratinocyte terminal differentiation is the apoptosis-like process that generates corneocytes for the desquaming

the pathogenesis of psoriasis, including IFN-γ and TNF-α.

**3.1.4 Keratinocytes and IL-18** 

plaque development remains to be elucidated.

**3.1.5 Keratinocytes and IL-20** 

TNF-α is a key pro-inflammatory cytokine with an important pathogenetic role in psoriasis and psoriatic arthritis. The evidence includes further observations that a variety of anti-TNF-α approaches such as monoclonal antibodies and fusion proteins of soluble TNF-α receptors are effective therapies both in psoriasis and psoriatic arthritis. As for TNF-α itself, production of this cytokine is mainly attributed to immune cells (Lowes et al., 2007); however, it is noteworthy that keratinocytes are also able to elaborate TNF-α (Gottlieb at al., 2005). In psoriasis, the inflammatory response to TNF-α could be self-sustaining: activated dendritic cells are the major source of TNF-α in psoriasis lesions (Boyman et al., 2004) and at the same time TNF-α mRNA is induced in keratinocytes after TNF-α exposure (Gottlieb et al., 2005). Low level of TNF-α is present in the upper layer of the healthy epidermis, but its synthesis and release from keratinocytes are greatly augmented by injury, infection and UV irradiation. Of the two distinct cell-surface receptors for TNF-α, TNFR1 and TNFR2, keratinocytes mainly express TNFR1 (Kondo & Sauder, 1997). The binding of TNF-α to TNFR1 triggers a series of intracellular events resulting in the activation of transcription factors, including NF-KB, AP-1, CCAAT enhancer-binding protein-β, and others, which are responsible for the induction of genes important for diverse biological processes, including cell growth and death and immune, inflammatory, and stress responses (Banno et al., 2004). TNF-α activates the immune responses through inducing the production of additional signals, such as IL-1 and IL-8, transforming growth factor type-β (TGF-β) and ICAM-1. Psoriatic keratinocytes are also an important source of IL-7. Increased IL-7 levels were observed in both the psoriatic skin and serum of psoriatic patients (Bonifati et al., 1997; Pietrzak et al., 2008). However, no correlation between IL-7 levels and psoriasis area severity index (PASI) score was observed. In addition, IL-7 levels did not decrease after effective anti-psoriatic treatment, which suggests that this cytokine could not be regarded as a marker of the disease activity. IL-7 is a pleiotropic cytokine playing an essential role in the development and differentiation of T cells. IL-7 regulates survival, proliferation and cytotoxicity of maturation of T cells at the periphery. Furthermore, IL-7 together with IL-2 and IL-12, can induce the synthesis of IFN-γ, while, in turn, IFN-γ induces IL-7 secretion by keratinocytes (Ariizumi et al., 1995). In psoriasis IL-7 seems to play a key role in driving

Pathogenesis of Psoriasis: The Role of Pro-Inflammatory Cytokines Produced by Keratinocytes 19

The typical erythema of psoriatic lesions is due to the increased, dilated, and tortuous capillaries that extend between the epidermal columns protruding into the dermis. The formation of new blood vessels starts with early psoriatic changes and disappears with disease clearance. Several angiogenic mediators like VEGF, hypoxia inducible factors, angiopoietins and pro-angiogenic cytokines, such as TNF-α, IL-8 and IL- 17, are involved in psoriasis development (Heidenreich et al., 2009). Interestingly, already in uninvolved, nonlesional skin significant over-expression of several VEGF isoforms was observed in patients as compared to healthy skin of normal volunteers (Henno et al., 2009). These findings suggest that angiogenesis is also one of the key features in the pathogenesis of psoriasis and various recent studies focused on the identification and role of pro-angiogenic mediators in psoriatic skin. In general, angiogenesis is tightly regulated by a balance between pro- and anti-angiogenic mediators (Heidenreich et al., 2009). VEGF, hypoxia-inducible factor-1α (HIF-1α), TNF-α, IL-8 and angiopoietins are considered to be the main players responsible for the increased vessel formation in psoriasis (Creamer et al., 2002; Heidenreich et al., 2008). Interestingly, several small molecules as well as modern biologics used for systemic therapy of psoriasis have been shown to provide not only immune regulatory effects but also influence endothelial cell biology (Heidenreich et al., 2008). Thus, direct targeting of angiogenesis could help both to dissect psoriasis pathogenesis and to develop new therapeutic strategies for psoriasis treatment by blocking angiogenic pathways driving cutaneous inflammation. Strongly increased production of VEGF by keratinocytes has been found in psoriasis (Detmar et al., 1994). Furthermore, over-expression of VEGF in the epidermis of mice triggered sub-epidermal angiogenesis and increased leukocyte adhesion to these vessels (Detmar et al., 1998), and later in life, these animals develop hyperkeratotic skin lesions with a resemblance to psoriasis (Xia et al., 2003). VEGF signaling often represents a critical rate-limiting step in physiological angiogenesis (Ferrara et al., 2003). Under physiological conditions, VEGF promotes growth of endothelial cells (ECs) derived from arteries, veins and lymphatic vessels. VEGF delivery also induces lymphoangiogenesis in mice and it is known to be a survival factor for endothelial cells both in vitro and in vivo. However, VEGF is also known as a vascular permeability factor, based on its ability to induce vascular leakage. In the meantime it is well established that such permeability enhancing activity underlies significant roles of this molecule in inflammation and other pathological circumstances (Ferrara et al., 2003). Besides its potential role in causing aberrant angiogenesis and vascular leakage in the upper dermis, VEGF may also contribute to keratinocyte proliferation and epidermal barrier homeostasis (Elias et al., 2008; Heidenreich et al., 2009). In psoriatic skin, the VEGF receptors VEGFR-1 and -2 are detectable and functional in keratinocytes (Man et al., 2006). As VEGF is secreted by keratinocytes and induces VEGFR expression in the same cells, VEGF may also contribute to keratinocyte hyperproliferation in psoriasis in an autocrine manner. This could be relevant when psoriasis is triggered by external injury (Koebner phenomenon) and interestingly disruption of the epidermal barrier homeostasis induces VEGF expression (Elias et al., 2008). Further evidence for a role of VEGF in keratinocyte proliferation comes from transgenic mice deficient in epidermal VEGF: these animals have delayed permeability barrier recovery after acute perturbation, decreased density of dermal blood vessels and lack epidermal hyperplasia as well as angiogenesis in response to sustained barrier disruption (Elias et al., 2008). Thus, physiological production of VEGF contributes to normal proliferation,

**3.3.1 Keratinocytes and VEGF** 

reciprocal interactions between epithelial cells (keratinocytes) and T-lymphocytes. The concomitant T-lymphocyte activation may be dependent on IL-7, and therefore the subsequent events driving toward the clinical expression and persistence of psoriasis may be IL-7 mediated (Bonifati et al., 1997). All these findings suggest an involvement of IL-7 in psoriasis, although further studies are warranted to elucidate the exact role of this molecule in the cytokine network of psoriasis pathogenesis.
